WO2006138366A2 - Protein markers - Google Patents

Protein markers Download PDF

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Publication number
WO2006138366A2
WO2006138366A2 PCT/US2006/023153 US2006023153W WO2006138366A2 WO 2006138366 A2 WO2006138366 A2 WO 2006138366A2 US 2006023153 W US2006023153 W US 2006023153W WO 2006138366 A2 WO2006138366 A2 WO 2006138366A2
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Prior art keywords
protein
dye
dyes
marker
molecular weight
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PCT/US2006/023153
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French (fr)
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WO2006138366A3 (en
Inventor
Dong Ma
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New England Biolabs, Inc.
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Publication of WO2006138366A2 publication Critical patent/WO2006138366A2/en
Publication of WO2006138366A3 publication Critical patent/WO2006138366A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • a protein marker which includes a protein covalently bound to: (i) two or more dyes; (ii) one or more dyes in a mixture with one or more non-dye protein-binding molecules; or (iii) two or more non-dye protein-binding molecules.
  • Competitive binding of the dye(s) and/or non-dye protein-binding molecules to the protein of a predetermined molecular weight results in a change in molecular weight and/or pi and optionally a change in color.
  • Examples of a dye in (i) or (ii) include a dye with a sulfone group such as a Remazol dye, a reactive chlorine such as monochlorotriazine or l-chloro-2,4-dinitrobenzene and an isothiocyanate such as Fluorescein-5-isothiocyanate (FITC) or Tetramethylrhodamine-5-isothiocyanate (TRITC).
  • the non-dye protein-binding molecule has a sulfone group such as Sulfo-NHS-LC-Biotin.
  • a protein marker includes two or more dyes that bind to the protein in a predetermined ratio, or one or more dyes and a non-dye protein-binding molecule that bind to the protein in a predetermined ratio, or two or more non-dye protein- binding molecules that bind to the protein in a predetermined ratio, so as to alter the molecular weight, pi and/or color of the protein as a function of the ratio of dyes and/or non-dye protein-binding molecules in the mixture.
  • the protein marker is characterized by a molecular weight that is related to the composition of the mixture of two or more dyes. This molecular weight may be greater than the molecular weight of the protein conjugated to one dye in the mixture but less than the molecular weight of the protein marker bound with another dye in the mixture. Similarly, the molecular weight of a protein marker may be greater when bound to at least one dye and one non-dye protein-binding molecule compared with binding to a dye or to a non-dye protein-binding molecule only.
  • the molecular weight of the protein marker can be varied according to the amount of the various non-dye protein- binding molecules where each molecule has an effect on the molecular weight of the protein.
  • the protein marker is a member of a set of protein markers in a ladder wherein each member of the set includes a protein with a distinct size and/or pi different from other members of the set and the set of markers has at least one color or alternatively two or more colors or alternatively three or more colors.
  • the mixture may contain a fluorescent dye and optionally a non-dye protein-binding molecule.
  • a method is provided of making a protein having a desired size that includes: (a) preparing a mixture of dyes or a mixture of at least one dye and a second non-dye protein-binding molecule; and (b) adding the dye mixture to a protein having a pre-selected molecular weight to form the protein marker having a desired size.
  • a method for making a ladder from proteins having altered molecular weights or pi that includes: (a) selecting one or more amino group-binding chemical reagents; and (b) mixing the one or more chemical reagents with a protein to form a size ladder or iso-electronic fusing marker ladder capable of being visualized on a gel by means of staining the gel.
  • the chemical reagent includes at least one of Methyl Vinyl Sulfone and Sulfo-NHS-LC-Biotin.
  • a ladder is formed from protein markers having predetermined molecular weights or pi where the protein markers are covalently bound to one or more amino group-binding chemical reagents to form a size ladder or iso- electronic fusing marker ladder, the ladder being capable of being visualized on a gel by means of staining.
  • amino group-binding chemical reagents are Methyl Vinyl Sulfone and Sulfo-NHS- LC-Biotin.
  • a detection means for detecting an assay endpoint, that includes linking by for example, intein-mediated ligation, a first protein to a first dye having a first color and a second protein linked to a second dye having a second color such that when the first protein becomes linked to the second protein, the first dye and the second dye produce a third color.
  • Figure 1 shows a set of pre-stained ladders as examples of those ladders that can be made using the present methods.
  • Lane 1 is blue pre-stained ladder
  • lane 2 is a Tri-color pre-stained protein ladder where band 5 (60 Kd) is orange and band 10 (25 Kd) is red, while the remainders are blue.
  • Lane 3 is a green pre-stained ladder
  • Lane 4 is a Tri-color green-, red- and orange-stained ladder where the orange band is 60 Kd and the red band is 25 Kd while the remainder are green.
  • Lane 5 is a rainbow-colored pre-stained ladder with red, orange, yellow, green, light blue, dark blue and purple bands.
  • Lane 6 is a multi-colored pre-stained protein ladder with each band being a different color.
  • Lane 7 is a blue and biotinylated protein ladder for two applications on either a pre- stained marker or a western blot marker using biotin-streptavidin function.
  • Lane 8 is a ladder that appears blue under natural light and green/red under ultraviolet light.
  • Figure 2 shows a 4-20% SDS-polyacrylamide gel (Tris-glycine buffer) in which the various dye mixtures added to maltose-binding protein (MBP) (Cone. 2 mg/ml) by incubating at 55 0 C for 4 hours. 10 ⁇ l of MBP (Cone. 2 mg/ml) is used throughout. The concentration of each Remazol dye is 100 mg/ml. Lanes 2-11 are as follows: Lane 1 is the NEB pre-stained protein marker (NEB, Cat. P7708, New England Biolabs, Inc., Ipswich, MA).
  • NEB NEB pre-stained protein marker
  • Lane 2 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) (Aldrich Cat. 109932, Sigma-Aldrich, St. Louis, MO) + 5 ⁇ l of Remazol Brilliant Blue R (Sigma, Cat. R8001, Sigma- Aldrich, St. Louis, MO).
  • Lane 3 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) + 5 ⁇ l of Remazol Brilliant Red BB (RR21) (Sigma-Aldrich, St. Louis, MO).
  • Lane 4 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) + 5 ⁇ l of Remazol Brilliant Orange 3R (Aldrich, Cat. 306509, Sigma-Aldrich, St. Louis, MO).
  • Lane 5 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) + 5 ⁇ l of Remazol yellow GR (Aldrich, R318914, Sigma-Aldrich, St. Louis, MO).
  • Lane 6 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) + 5 ⁇ l of Remazol Brilliant Violet 5R (Sigma, Cat. R6006, Sigma-Aldrich, St. Louis, MO).
  • Lane 7 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) + 2 ⁇ l of Remazol Brilliant Blue R+ 3 ⁇ l of Remazol Yellow GR (Sigma-Aldrich, St. Louis, MO).
  • Lane 8 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) + l ⁇ l of Remazol Brilliant Blue R+ 4 ⁇ l of Remazol Yellow GR (Sigma-Aldrich, St. Louis, MO).
  • Lane 9 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) + 3 ⁇ l of Remazol Yellow GR + 2 ⁇ l of Remazol Brilliant Red BB (RR21) (Sigma-Aldrich, St. Louis, MO).
  • Lane 10 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) + 3 ⁇ l of Remazol Yellow GR + 2 ⁇ l of Remazol Brilliant Orange 3R (Sigma-Aldrich, St. Louis, MO).
  • Lane 11 shows MBP added to a mixture of 5 ⁇ l of N-ethyl morpholine (100%) + 2 ⁇ l of Remazol Brilliant Violet 5R+ 3 ⁇ l of Remazol Yellow GR (Sigma-Aldrich, St. Louis, MO).
  • Figure 3 shows the results of various dye mixtures added to MBP-truncated beta-gal (MW: 60kd, Cone. 2 mg/ml) and incubated at 55 0 C for 4 hours.
  • Lane 1 is the NEB pre-stained protein marker (NEB, Cat. P7708, New England Biolabs, Inc., Ipswich, MA). 10 ⁇ l of protein is used throughout. Every mixture of dye includes 5 ⁇ l of N- ethyl morpholine (100%)(Aldrich, Cat 109932, Sigma-Aldrich, St. Louis, MO). The concentration of each Remazol dye is 100mg/ml.
  • Lanes 2-11 show protein markers (conjugates) in which a mixture of dyes including N-ethyl morpholine and at least one Remazol dye are added to MBP truncated beta-gal. The lanes are as follows:
  • Lane 2 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + 5 ⁇ l of Remazol Brilliant Blue R (Sigma, Cat. R8001, Sigma-Aldrich, St. Louis, MO).
  • Lane 3 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + 4 ⁇ l of Remazol Brilliant Blue R + l ⁇ l of Remazol Brilliant Violet 5R (Sigma, Cat. R6006, Sigma-Aldrich, St. Louis, MO).
  • Lane 4 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + 3 ⁇ l of Remazol Brilliant Blue R+ 2 ⁇ l of Remazol Brilliant Violet 5R.
  • Lane 5 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + 2 ⁇ l of Remazol Brilliant Blue R+ 3 ⁇ l of Remazol Brilliant Violet 5R.
  • Lane 6 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + l ⁇ l of Remazol Brilliant Blue R+ 4 ⁇ l of Remazol Brilliant Violet 5R.
  • Lane 7 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + 5 ⁇ l of Remazol Brilliant Violet 5R.
  • Lane 8 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + 4 ⁇ l of Remazol Brilliant Violet 5R and 1 ⁇ l of Remazol Yellow GR (Aldrich Cat. R318914).
  • Lane 9 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + 3 ⁇ l of Remazol Brilliant Violet 5R+ 2 ⁇ l of Remazol Yellow GR.
  • Lane 10 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + 2 ⁇ l of Remazol Brilliant Violet 5R+ 3 ⁇ l Remazol Yellow GR.
  • Lane 11 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + l ⁇ l of Remazol Brilliant Violet 5R+ 4 ⁇ l Remazol Yellow GR.
  • Lane 12 is a protein marker in which the dye mixture added to the protein is 5 ⁇ l of N-ethyl morpholine (100%) + 5 ⁇ l of Remazol yellow GR.
  • Figure 4 shows the same mixtures of dyes used in Figure 3 added to an MBP (MW: 40 Kd) at 2 mg/ml.
  • Figure 5 shows the same mixtures of dyes used in Figure 3 added to E. coli RNaseIII protein (MW: 25 Kd) at 2 mg/ml.
  • Figure 6 shows the same mixtures of dyes used in Figure 3 added to Myoglobin protein (Sigma Cat. M0630, Sigma-Aldrich, St. Louis, MO) at Cone. 2 mg/ml.
  • Figure 7 shows the results of various dye mixtures added to MBP-truncated beta-gal (MW: ⁇ 60 Kd, Concentration - 2 mg/ml) and incubated at 55° C for 4 hours.
  • Lane 1 is the Bio-Rad precision plus protein standards All Blue (Bio-Rad, Cat. 161-0373, Bio-Rad Laboratories, Hercules, CA)
  • lane 2 is the Bio-Rad precision plus protein standards dual color (Bio-Rad, Cat. 161-0374)
  • lanes 3 and 12 are NEB pre-stained protein markers (NEB, Cat. P7708, New England Biolabs, Inc., Ipswich, MA). 10 ⁇ l of protein is used throughout.
  • Every mixture of dye includes 5 ⁇ l of N-ethyl morpholine (100%).
  • the concentration of each Remazol dye is 100 mg/ml.
  • Lanes 4-11 show protein markers in which a mixture of dyes including 5 ⁇ l of N-ethyl morpholine and at least one Remazol dye are added to MBP truncated beta-gal.
  • the Remazol dye amounts in lanes 4-11 are as follows: Lane 4: 3 ⁇ l of Remazol Yellow GR (Aldrich Cat. R318914), 2 ⁇ l of Remazol Brilliant Orange 3R (Aldrich Cat. 306509);
  • Lane 5 3.5 ⁇ l of Remazol Yellow GR, 1.5 ⁇ l of Remazol Brilliant Orange 3R.
  • Lane 6 4.5 ⁇ l of Remazol Yellow GR, 0.5 ⁇ l of Remazol Brilliant Orange 3R.
  • Lane 7 2.5 ⁇ l of Remazol Brilliant Blue R (Sigma Cat. 8001), 2.5 ⁇ l of Remazol Yellow GR.
  • Lane 8 2.5 ⁇ l o Remazol Brilliant Blue R, 2.5 ⁇ l of Remazol Brilliant Violet 5R (Sigma Cat. R6006). Lane 9: 1.5 ⁇ l of Remazol Brilliant Blue R, 3.5 ⁇ l of Remazol
  • Lane 10 0.5 ⁇ l of Remazol Brilliant Blue R, 4.5 ⁇ l of Remazol Brilliant Violet 5R.
  • Lane 11 4 ⁇ l of Remazol Brilliant Blue R, l ⁇ l of Remazol Brilliant Violet 5R.
  • Figure 8 shows the results of mixtures of dyes as prepared in Figure 7 but added to E. coli RNAse III protein (MW: 25 Kd) at 2 mg/ml.
  • Figure 9 shows an assay for selecting a mixture of dyes for generating a desired protein ladder.
  • Various dye mixtures were added to MBP-truncated beta-gal (MW: ⁇ 60kd) at 2 mg/ml and incubated at 55 0 C for 4 hours. 10 ⁇ l of protein are used throughout. Every mixture of dye included 5 ⁇ l of N-ethyl morpholine (100%). The concentration of each Remazol dye is 100mg/ml.
  • Lanes 2-12 show protein markers in which a mixture of dyes including 5 ⁇ l N- ethyl morpholine and at least one Remazol dye was added to MBP truncated beta-gal : Lane 1 is an NEB pre-stained protein marker (NEB, Cat.
  • Lane 2 5 ⁇ l of Remazol Brilliant Blue R (Sigma, Cat. 8001).
  • Lane 3 5 ⁇ l of Reactive Blue Remazol Turquoise P (Aldrich, Cat. R309052). Lane 4: 5 ⁇ l of Remazol Red F3B (Aldrich, Cat. S478997).
  • Lane 5 5 ⁇ l of Remazol Red 3B (Aldrich, Cat. R318906).
  • Lane 6 5 ⁇ l of Remazol Brilliant Red 5 BA (Aldrich, Cat. R323705).
  • Lane 7 5 ⁇ l of Remazol Brilliant Green 5 GA (Aldrich, Cat. S466662).
  • Lane 8 5 ⁇ l of Remazol Green B (Aldrich, Cat. S466700).
  • Lane 9 5 ⁇ l of Remazol Brown GR (Aldrich, Cat. R323691).
  • Lane 10 5 ⁇ l of Remazol Brilliant Violet 5R (Aldrich, Cat. 468959). Lane 11 : 5 ⁇ l of Remazol Red Violet R (Aldrich, Cat. 468940).
  • Lane 12 Remazol Brilliant Orange 3R (Aldrich, Cat. 306509).
  • Figure 10 shows an assay for selecting a mixture of dyes for generating a desired protein ladder.
  • Various dye mixtures were added to MBP-truncated beta-gal (MW: ⁇ 60kd) at Cone. 2 mg/ml and incubated at 55° C for 4 hours. 10 ⁇ l of protein were used throughout. Every mixture of dye includes 5 ⁇ l of N-ethyl morpholine (100%). The concentration of each Remazol dye is 100 mg/ml.
  • Lanes 2-12 show protein markers in which a mixture of dyes including 5 ⁇ l N-ethyl morpholine and at least one Remazol dye is added to MBP truncated beta-gal protein.
  • Lane 1 is the precision plus protein standards dual color (Bio- Rad, Cat 161-0374).
  • Lane 2 5 ⁇ l of Remazol Brilliant Blue R (Sigma, Cat. 8001). Lane 3: 5 ⁇ l of Remazol Red F3B (Aldrich, Cat. S478997).
  • Lane 4 5 ⁇ l of Remazol Red 3B (Aldrich, Cat. R318906). Lane 5: 5 ⁇ l of Remazol Brilliant Red 5 BA (Aldrich, Cat. R323705).
  • Lane 6 5 ⁇ l of Remazol Brilliant Orange 3R (Aldrich, Cat. 306509).
  • Lane 7 5 ⁇ l of Remazol Brilliant Violet 5R (Sigma, Cat. R6006).
  • Lane 8 5 ⁇ l of Remazol Brilliant Violet 5R (Aldrich, Cat. 468959). Lane 9: 5 ⁇ l of Remazol Red Violet R (Aldrich, Cat. 468940).
  • Lane 10 5 ⁇ l of Remazol Brown GR (Aldrich, Cat. 323691). Lane 11: 5 ⁇ l of Remazol Yellow GR (Aldrich, Cat. R318914). Lane 12: 5 ⁇ l of Remazol Yellow FG (Aldrich, Cat. S472301).
  • FIG. 11 shows the same mixture of dyes as described in
  • Figure 10 added to MBP (MW: 40 Kd, 2 mg/ml) to form a conjugate.
  • Figure 12 shows the same mixture of dyes as described in Figure 10 added to lysozyme (Sigma Cat. L4631) at 2 mg/ml to form a protein marker.
  • Figure 13 shows the same mixture of dyes as described in Figure 10 added to myoglobin protein (Sigma, Cat. M0630) to form a protein marker.
  • Figure 14 shows how a protein conjugate runs on a 4-20% SDS-polyacrylamide gel (Tris-Glycine buffer) when Remazol Brilliant Blue R (Sigma, Cat. R8001) at 100 mg/ml is mixed with Methyl Vinyl Sulfone (Aldrich Cat. 247197, 5 mg/ml) in various amounts and then added to 10 ⁇ l of MBP truncated beta-gal (2 mg/ml) in Lane 2-7, 10 ⁇ l of MBP (2mg/ml) in lanes 8-13 and the mixture are incubated at 55 0 C for 4 hours.
  • Coomassie Blue R-250 (Bio- Rad Cat. No. 161-0400) is needed to stain the gel : Lane 1 : NEB pre-stained marker (NEB, Cat P7708, New
  • Lane 2 5 ⁇ l of Remazol Brilliant Blue R (Sigma, Cat. R8001). Lane 3: 3.75 ⁇ l of Remazol Brilliant Blue R, 1.25 ⁇ l of Methyl Vinyl Sulfone. Lane 4: 2.5 ⁇ l of Remazol Brilliant Blue R, 2.5 ⁇ l of Methyl
  • Lane 5 Remazol Brilliant Blue R (1.25 ⁇ l), 3.75 ⁇ l of Methyl Vinyl Sulfone.
  • Lane 6 5 ⁇ l of Methyl Vinyl Sulfone. Lane 7: MBP-truncated beta-gal only.
  • Lane 8 5 ⁇ l of Remazol Brilliant Blue R.
  • Lane 9 3.75 ⁇ l of Remazol Brilliant Blue R, 1.25 ⁇ l of Methyl Vinyl Sulfone.
  • Lane 10 2.5 ⁇ l of Remazol Brilliant Blue R, 2.5 ⁇ i of Methyl Vinyl Sulfone.
  • Lane 11 1.25 ⁇ l of Remazol Brilliant Blue R, 3.75 ⁇ l of Methyl Vinyl Sulfone.
  • Lane 13 MBP only.
  • Figure 15 shows mixtures as described in Figure 14 conjugated to the same proteins as in Figure 14 except that the Methyl Vinyl Sulfone is substituted by Sulfo-NHS-LC-Biotin (Pierce Biotechnology, Inc. Cat. 21335) at 5 mg/ml.
  • Figures 16-1 (A, B, C, D, E, F) - Figures 16-2 (G, H, I) show the chemical structures of two Remazol dyes (A and B); Methyl Vinyl Sulfone and Sulfo-NHS-LC-Biotin (C and D); two isothiocyanates, TRITC and FITC (E and F), which are capable of reacting with a primary or a secondary amino group on protein; and l-Chloro-2,4-dinitrobenzene, Monochloro-s-triazine, Tris (2- carboxyethyl) phosphine hydrochloride (G, H and I), which react with an amino group through a chlorine ion.
  • a protein ladder having bands spaced at predetermined intervals has been solved here by a novel approach in which a protein is covalently bound to one or more dyes and/or optionally one or more non-dye protein-binding molecules.
  • Marker ladders can be readily assembled as desired using a single protein or different proteins and a series of combinations of dyes and/or non-dye protein-binding molecules. Two or more dyes mixed at various ratios are covalently bound to the protein sample to form a sharp band on a gel having a size that differs from the non-dyed protein. When different sized proteins are treated thus, a precise ladder can be constructed (see Figure 1).
  • a pre-stained protein band with a size of 50 Kd can be made from an unstained protein having a size of 37 Kd and two Remazol dyes mixed together in a predetermined ratio such as Remazol Brilliant Blue R at 31% and Remazol Brilliant Yellow at 69% of the mixture to the protein).
  • the dye mixture competitively binds to the reactive amino ligand on the protein. Changing the concentration of dyes in a mixture determines the outcome of the competitive binding reaction.
  • the ratio of dyes bound to the protein affects the size of the protein band in a predictable manner and also its color. The colors may span the entire spectrum of light according to the ratio of different dyes used. Examples of the wide range of colors that might be employed in a protein ladder are provided in the Figures. Dyes that produce a fluorescent signal detectable under ultraviolet light may be used in the mixture. Other dyes may be detectable using X-rays.
  • the use of mixtures of dyes as described herein provides a means to make a desired colored protein by staining the protein with different dyes such as Remazol dyes at selected ratios (see Figures 2-13).
  • the protein can be stained with a Remazol Brilliant Blue R and a Remazol Brilliant Yellow at a ratio of 1: 1.
  • the type of dyes and their combinations for any particular protein are selected to avoid fuzzy bands.
  • the MBP protein marker in Lane 9 is relatively fuzzy compared to the tight band in Lane 8. The sharpest bands occur in lanes 5-8 where lanes 5-6 are a single dye while lanes 7-8 are a mixture of 2 dyes.
  • the position of the protein bands can be modified not only by mixing two or more dyes but also by adding to the mixture or to one dye and a non-dye protein-binding molecule that bind to the protein competitively.
  • the non-dye protein-binding molecule can include a biological macromolecule such as a modified protein or a chemical reagent.
  • the ladder may be a single color.
  • the protein can be stained with a Remazol reactive dye (e.g. Remazol Brilliant Blue R) plus Methyl Vinyl Sulfone, which is a chemical reagent having a similar reactive group to the Remazol dyes but without the color-inducing aromatic rings (see Figure 14).
  • Remazol reactive dye e.g. Remazol Brilliant Blue R
  • Methyl Vinyl Sulfone which is a chemical reagent having a similar reactive group to the Remazol dyes but without the color-inducing aromatic rings (see Figure 14).
  • a protein ladder both in natural light and on an X-ray film.
  • This may be useful in Western Blotting for example. This can be achieved, for example, by staining a protein with varying ratios of a Remazol dye and Sulfo-NHS-LC Biotin (see Figure 15) providing a suitable color and size on the gel.
  • a Remazol dye and a fluorescent reagent e.g. FITC or TRITC
  • FITC or TRITC can be used to stain a protein at a ratio suitable for generating a particular size band on a gel and for visualizing this protein both in natural light and under ultraviolet light.
  • An advantage of having a protein ladder formed from proteins of different sizes having different colors is that it is immediately possible to conclude how well the gel is running and how much it has already run by noting the position of a band of known size corresponding to a particular color.
  • Example 1 Preparation of a third-colored protein by staining with two different Remazol dyes at a certain ratio and preparation of a green-colored protein band from staining with blue and yellow dyes.
  • Protein staining A mixture including 500 ⁇ l of N- Ethylmorpholine (Aldrich, Cat. 109932, Sigma-Aldrich, St. Louis, MO), 1 ml of a pretreated MBP, 200 ⁇ l of Blue dye solution and 300 ⁇ l of Yellow dye solution was incubated at 55° C for 4 hours or 37° C for overnight (see Lane 7 in Figure 2). To terminate this reaction, L- Lysine (Sigma, Cat. L5501, Sigma-Aldrich, St. Louis, MO) was added to 10 mM in the dye mixture. A 2 ⁇ l aliquot of reaction mixture was analyzed on a Novex 4-20% Tris-glycine gel (Invitrogen, Cat.
  • reaction mixture was dialyzed in a buffer (10 mM Tris-HCI pH 7.0, 50 mM NaCI) until all excess dyes were removed.
  • the colored protein was stored in a -20° C freezer.
  • Example 2 Preparation of two light violet-colored proteins by staining with a mixture of two Remazol dyes at a pre-selected ratio.
  • Remazol Brilliant Violet 5R (Sigma, Cat. R6006) and 100 mg of Remazol Brilliant Yellow GR (Aldrich, Cat. R318914) were weighed and separately dissolved in 1 ml of DH 2 O to make a Violet dye solution (100 mg/ml) and a Yellow dye solution (100 mg/ml).
  • a purified MBP (40 kd, 2 mg/ml in 6OmM Carborate buffer, pH 9.6) was pretreated by adding SDS to 0.2% and TCEP (Sigma, Cat. C4706) to 10 mM. The preparation was incubated at 95° C for 3 min.
  • prestained MBP of size 70Kd For making prestained MBP of size 70Kd, a mixture including 500 ⁇ l of 100%N-Ethylmorpholine (Aldrich, Cat. 109932), 1 ml of a pretreated MBP, 400 ⁇ l of Violet dye solution and 100 ⁇ l of Yellow dye solution (the ratio of violet dye: yellow dye is 4:1) were incubated at 55 0 C for 4 hours or 37° C for overnight. For making a prestained MBP having a size of 50Kd, the ratio of Violet dye: Yellow dye used was 2:3 instead of 4: 1 using the same conditions as described above. To terminate the reaction, L-Lysine was added (Sigma, Cat. L5501) to 10 mM in this dye mixture.
  • reaction mixture An aliquot of 2 ⁇ l of reaction mixture was tested on a Novex 4-20 % Tris-glycine gel (Invitrogen, Cat. EC60255) to see if reaction worked. To remove the free dyes, the reaction mixture was dialyzed against a buffer (10 mM Tris-HCI pH 7.0, 50 mM NaCI) until all excess dyes were removed. The colored protein was stored in a -20° C freezer.

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Abstract

A protein marker and a ladder that contains a series of different protein markers are described. Each protein marker is a product of a protein covalently bound to: two or more dyes; one or more dyes and a non-dye protein-binding molecule; or two or more non-dye protein-binding molecules; having a predetermined molecular weight, pi and/or color.

Description

Protein Markers
BACKGROUND
Forming a ladder of detectable evenly spaced protein size markers for gel electrophoresis has been a problem until now because proteins do not naturally occur in sizes suitable for forming a ladder. Moreover when dyes are covalently bound to the component proteins or peptides, the position of the protein or peptide of a particular size in the ladder is altered and the desired spacing between different markers may be lost. Other factors also alter the relative position of different proteins in a ladder such as the gel itself, the buffer used to run the gel and the extent of colored dye bound to a particular protein band. For example, a protein having a size of 35 Kd may, when stained, run on a gel as if it had a size of 53 Kd.
Designing a protein size ladder using existing protocols is complicated still further by staining a majority of proteins in a ladder with one dye and one or two proteins in the ladder with an alternate dye. A protein marker stained with an alternate dye in a size ladder has been used to determine the extent of migration of a protein of unknown size on a gel and also to determine the quality of the gel (see for example US 4,507,233, 5,449,758 and 6,703,484). However, the alternate dye-protein complex may disrupt the spacing of component bands in the protein ladder because of different migration patterns owing to size and charge of the conjugate compared with that of the first dye. SUMMARY
In an embodiment of the invention, a protein marker is described which includes a protein covalently bound to: (i) two or more dyes; (ii) one or more dyes in a mixture with one or more non-dye protein-binding molecules; or (iii) two or more non-dye protein-binding molecules. Competitive binding of the dye(s) and/or non-dye protein-binding molecules to the protein of a predetermined molecular weight results in a change in molecular weight and/or pi and optionally a change in color.
Examples of a dye in (i) or (ii) include a dye with a sulfone group such as a Remazol dye, a reactive chlorine such as monochlorotriazine or l-chloro-2,4-dinitrobenzene and an isothiocyanate such as Fluorescein-5-isothiocyanate (FITC) or Tetramethylrhodamine-5-isothiocyanate (TRITC). In another example, the non-dye protein-binding molecule has a sulfone group such as Sulfo-NHS-LC-Biotin.
In an embodiment, a protein marker includes two or more dyes that bind to the protein in a predetermined ratio, or one or more dyes and a non-dye protein-binding molecule that bind to the protein in a predetermined ratio, or two or more non-dye protein- binding molecules that bind to the protein in a predetermined ratio, so as to alter the molecular weight, pi and/or color of the protein as a function of the ratio of dyes and/or non-dye protein-binding molecules in the mixture.
In one embodiment, the protein marker is characterized by a molecular weight that is related to the composition of the mixture of two or more dyes. This molecular weight may be greater than the molecular weight of the protein conjugated to one dye in the mixture but less than the molecular weight of the protein marker bound with another dye in the mixture. Similarly, the molecular weight of a protein marker may be greater when bound to at least one dye and one non-dye protein-binding molecule compared with binding to a dye or to a non-dye protein-binding molecule only.
Where a mixture of two or more non-dye protein-binding molecules are added to a protein in the absence of a dye to form a protein marker, the molecular weight of the protein marker can be varied according to the amount of the various non-dye protein- binding molecules where each molecule has an effect on the molecular weight of the protein.
In an embodiment of the invention, the two or more dyes in
(i) or (ii) are selected so that the dyes competitively bind the protein to generate a protein marker having a desired color that is different from the color of the dyes before binding to the protein.
In an additional embodiment of the invention, the protein marker is a member of a set of protein markers in a ladder wherein each member of the set includes a protein with a distinct size and/or pi different from other members of the set and the set of markers has at least one color or alternatively two or more colors or alternatively three or more colors. Alternatively or additionally, the mixture may contain a fluorescent dye and optionally a non-dye protein-binding molecule.
In an additional embodiment of the invention, a method is provided of making a protein having a desired size that includes: (a) preparing a mixture of dyes or a mixture of at least one dye and a second non-dye protein-binding molecule; and (b) adding the dye mixture to a protein having a pre-selected molecular weight to form the protein marker having a desired size.
In an additional embodiment, a method is provided for making a ladder from proteins having altered molecular weights or pi that includes: (a) selecting one or more amino group-binding chemical reagents; and (b) mixing the one or more chemical reagents with a protein to form a size ladder or iso-electronic fusing marker ladder capable of being visualized on a gel by means of staining the gel. In one example, the chemical reagent includes at least one of Methyl Vinyl Sulfone and Sulfo-NHS-LC-Biotin.
In another embodiment of the invention, a ladder is formed from protein markers having predetermined molecular weights or pi where the protein markers are covalently bound to one or more amino group-binding chemical reagents to form a size ladder or iso- electronic fusing marker ladder, the ladder being capable of being visualized on a gel by means of staining. Examples of amino group- binding chemical reagents are Methyl Vinyl Sulfone and Sulfo-NHS- LC-Biotin.
In an embodiment of the invention, a detection means is provided for detecting an assay endpoint, that includes linking by for example, intein-mediated ligation, a first protein to a first dye having a first color and a second protein linked to a second dye having a second color such that when the first protein becomes linked to the second protein, the first dye and the second dye produce a third color. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a set of pre-stained ladders as examples of those ladders that can be made using the present methods. Lane 1 is blue pre-stained ladder, lane 2 is a Tri-color pre-stained protein ladder where band 5 (60 Kd) is orange and band 10 (25 Kd) is red, while the remainders are blue. Lane 3 is a green pre-stained ladder and Lane 4 is a Tri-color green-, red- and orange-stained ladder where the orange band is 60 Kd and the red band is 25 Kd while the remainder are green. Lane 5 is a rainbow-colored pre-stained ladder with red, orange, yellow, green, light blue, dark blue and purple bands. Lane 6 is a multi-colored pre-stained protein ladder with each band being a different color. Lane 7 is a blue and biotinylated protein ladder for two applications on either a pre- stained marker or a western blot marker using biotin-streptavidin function. Lane 8 is a ladder that appears blue under natural light and green/red under ultraviolet light.
Figure 2 shows a 4-20% SDS-polyacrylamide gel (Tris-glycine buffer) in which the various dye mixtures added to maltose-binding protein (MBP) (Cone. 2 mg/ml) by incubating at 550C for 4 hours. 10 μl of MBP (Cone. 2 mg/ml) is used throughout. The concentration of each Remazol dye is 100 mg/ml. Lanes 2-11 are as follows: Lane 1 is the NEB pre-stained protein marker (NEB, Cat. P7708, New England Biolabs, Inc., Ipswich, MA).
Lane 2 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) (Aldrich Cat. 109932, Sigma-Aldrich, St. Louis, MO) + 5μl of Remazol Brilliant Blue R (Sigma, Cat. R8001, Sigma- Aldrich, St. Louis, MO). Lane 3 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) + 5μl of Remazol Brilliant Red BB (RR21) (Sigma-Aldrich, St. Louis, MO).
Lane 4 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) + 5μl of Remazol Brilliant Orange 3R (Aldrich, Cat. 306509, Sigma-Aldrich, St. Louis, MO).
Lane 5 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) + 5μl of Remazol yellow GR (Aldrich, R318914, Sigma-Aldrich, St. Louis, MO). Lane 6 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) + 5μl of Remazol Brilliant Violet 5R (Sigma, Cat. R6006, Sigma-Aldrich, St. Louis, MO).
Lane 7 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) + 2μl of Remazol Brilliant Blue R+ 3μl of Remazol Yellow GR (Sigma-Aldrich, St. Louis, MO).
Lane 8 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) + lμl of Remazol Brilliant Blue R+ 4μl of Remazol Yellow GR (Sigma-Aldrich, St. Louis, MO).
Lane 9 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) + 3μl of Remazol Yellow GR + 2μl of Remazol Brilliant Red BB (RR21) (Sigma-Aldrich, St. Louis, MO).
Lane 10 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) + 3μl of Remazol Yellow GR + 2μl of Remazol Brilliant Orange 3R (Sigma-Aldrich, St. Louis, MO). Lane 11 shows MBP added to a mixture of 5μl of N-ethyl morpholine (100%) + 2μl of Remazol Brilliant Violet 5R+ 3μl of Remazol Yellow GR (Sigma-Aldrich, St. Louis, MO).
Figure 3 shows the results of various dye mixtures added to MBP-truncated beta-gal (MW: 60kd, Cone. 2 mg/ml) and incubated at 550C for 4 hours. Lane 1 is the NEB pre-stained protein marker (NEB, Cat. P7708, New England Biolabs, Inc., Ipswich, MA). 10 μl of protein is used throughout. Every mixture of dye includes 5 μl of N- ethyl morpholine (100%)(Aldrich, Cat 109932, Sigma-Aldrich, St. Louis, MO). The concentration of each Remazol dye is 100mg/ml. Lanes 2-11 show protein markers (conjugates) in which a mixture of dyes including N-ethyl morpholine and at least one Remazol dye are added to MBP truncated beta-gal. The lanes are as follows:
Lane 2 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + 5μl of Remazol Brilliant Blue R (Sigma, Cat. R8001, Sigma-Aldrich, St. Louis, MO).
Lane 3 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + 4 μl of Remazol Brilliant Blue R + lμl of Remazol Brilliant Violet 5R (Sigma, Cat. R6006, Sigma-Aldrich, St. Louis, MO).
Lane 4 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + 3μl of Remazol Brilliant Blue R+ 2μl of Remazol Brilliant Violet 5R.
Lane 5 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + 2μl of Remazol Brilliant Blue R+ 3μl of Remazol Brilliant Violet 5R.
Lane 6 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + lμl of Remazol Brilliant Blue R+ 4μl of Remazol Brilliant Violet 5R. Lane 7 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + 5μl of Remazol Brilliant Violet 5R.
Lane 8 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + 4 μl of Remazol Brilliant Violet 5R and 1 μl of Remazol Yellow GR (Aldrich Cat. R318914).
Lane 9 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + 3μl of Remazol Brilliant Violet 5R+ 2μl of Remazol Yellow GR.
Lane 10 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + 2μl of Remazol Brilliant Violet 5R+ 3μl Remazol Yellow GR.
Lane 11 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + lμl of Remazol Brilliant Violet 5R+ 4μl Remazol Yellow GR.
Lane 12 is a protein marker in which the dye mixture added to the protein is 5 μl of N-ethyl morpholine (100%) + 5μl of Remazol yellow GR.
Figure 4 shows the same mixtures of dyes used in Figure 3 added to an MBP (MW: 40 Kd) at 2 mg/ml.
Figure 5 shows the same mixtures of dyes used in Figure 3 added to E. coli RNaseIII protein (MW: 25 Kd) at 2 mg/ml.
Figure 6 shows the same mixtures of dyes used in Figure 3 added to Myoglobin protein (Sigma Cat. M0630, Sigma-Aldrich, St. Louis, MO) at Cone. 2 mg/ml.
Figure 7 shows the results of various dye mixtures added to MBP-truncated beta-gal (MW: ~60 Kd, Concentration - 2 mg/ml) and incubated at 55° C for 4 hours. Lane 1 is the Bio-Rad precision plus protein standards All Blue (Bio-Rad, Cat. 161-0373, Bio-Rad Laboratories, Hercules, CA), lane 2 is the Bio-Rad precision plus protein standards dual color (Bio-Rad, Cat. 161-0374) and lanes 3 and 12 are NEB pre-stained protein markers (NEB, Cat. P7708, New England Biolabs, Inc., Ipswich, MA). 10 μl of protein is used throughout. Every mixture of dye includes 5 μl of N-ethyl morpholine (100%). The concentration of each Remazol dye is 100 mg/ml. Lanes 4-11 show protein markers in which a mixture of dyes including 5μl of N-ethyl morpholine and at least one Remazol dye are added to MBP truncated beta-gal. The Remazol dye amounts in lanes 4-11 are as follows: Lane 4: 3 μl of Remazol Yellow GR (Aldrich Cat. R318914), 2 μl of Remazol Brilliant Orange 3R (Aldrich Cat. 306509);
Lane 5: 3.5 μl of Remazol Yellow GR, 1.5 μl of Remazol Brilliant Orange 3R.
Lane 6: 4.5 μl of Remazol Yellow GR, 0.5 μl of Remazol Brilliant Orange 3R.
Lane 7: 2.5 μl of Remazol Brilliant Blue R (Sigma Cat. 8001), 2.5 μl of Remazol Yellow GR.
Lane 8: 2.5 μl o Remazol Brilliant Blue R, 2.5 μl of Remazol Brilliant Violet 5R (Sigma Cat. R6006). Lane 9: 1.5 μl of Remazol Brilliant Blue R, 3.5 μl of Remazol
Brilliant Violet 5R.
Lane 10: 0.5 μl of Remazol Brilliant Blue R, 4.5 μl of Remazol Brilliant Violet 5R.
Lane 11 : 4 μl of Remazol Brilliant Blue R, lμl of Remazol Brilliant Violet 5R.
Figure 8 shows the results of mixtures of dyes as prepared in Figure 7 but added to E. coli RNAse III protein (MW: 25 Kd) at 2 mg/ml. Figure 9 shows an assay for selecting a mixture of dyes for generating a desired protein ladder. Various dye mixtures were added to MBP-truncated beta-gal (MW: ~60kd) at 2 mg/ml and incubated at 550 C for 4 hours. 10 μl of protein are used throughout. Every mixture of dye included 5 μl of N-ethyl morpholine (100%). The concentration of each Remazol dye is 100mg/ml. Lanes 2-12 show protein markers in which a mixture of dyes including 5μl N- ethyl morpholine and at least one Remazol dye was added to MBP truncated beta-gal : Lane 1 is an NEB pre-stained protein marker (NEB, Cat.
P7708, New England Biolabs, Inc., Ipswich, MA).
Lane 2: 5μl of Remazol Brilliant Blue R (Sigma, Cat. 8001).
Lane 3: 5μl of Reactive Blue Remazol Turquoise P (Aldrich, Cat. R309052). Lane 4: 5μl of Remazol Red F3B (Aldrich, Cat. S478997).
Lane 5: 5μl of Remazol Red 3B (Aldrich, Cat. R318906).
Lane 6: 5μl of Remazol Brilliant Red 5 BA (Aldrich, Cat. R323705).
Lane 7: 5μl of Remazol Brilliant Green 5 GA (Aldrich, Cat. S466662).
Lane 8: 5μl of Remazol Green B (Aldrich, Cat. S466700).
Lane 9: 5μl of Remazol Brown GR (Aldrich, Cat. R323691).
Lane 10: 5μl of Remazol Brilliant Violet 5R (Aldrich, Cat. 468959). Lane 11 : 5μl of Remazol Red Violet R (Aldrich, Cat. 468940).
Lane 12: Remazol Brilliant Orange 3R (Aldrich, Cat. 306509).
Figure 10 shows an assay for selecting a mixture of dyes for generating a desired protein ladder. Various dye mixtures were added to MBP-truncated beta-gal (MW: ~60kd) at Cone. 2 mg/ml and incubated at 55° C for 4 hours. 10μl of protein were used throughout. Every mixture of dye includes 5 μl of N-ethyl morpholine (100%). The concentration of each Remazol dye is 100 mg/ml. Lanes 2-12 show protein markers in which a mixture of dyes including 5μl N-ethyl morpholine and at least one Remazol dye is added to MBP truncated beta-gal protein.
Lane 1 is the precision plus protein standards dual color (Bio- Rad, Cat 161-0374).
Lane 2: 5μl of Remazol Brilliant Blue R (Sigma, Cat. 8001). Lane 3: 5μl of Remazol Red F3B (Aldrich, Cat. S478997).
Lane 4: 5μl of Remazol Red 3B (Aldrich, Cat. R318906). Lane 5: 5μl of Remazol Brilliant Red 5 BA (Aldrich, Cat. R323705).
Lane 6: 5μl of Remazol Brilliant Orange 3R (Aldrich, Cat. 306509).
Lane 7: 5μl of Remazol Brilliant Violet 5R (Sigma, Cat. R6006).
Lane 8: 5μl of Remazol Brilliant Violet 5R (Aldrich, Cat. 468959). Lane 9: 5μl of Remazol Red Violet R (Aldrich, Cat. 468940).
Lane 10: 5μl of Remazol Brown GR (Aldrich, Cat. 323691). Lane 11: 5μl of Remazol Yellow GR (Aldrich, Cat. R318914). Lane 12: 5μl of Remazol Yellow FG (Aldrich, Cat. S472301).
Figure 11 shows the same mixture of dyes as described in
Figure 10 added to MBP (MW: 40 Kd, 2 mg/ml) to form a conjugate.
Figure 12 shows the same mixture of dyes as described in Figure 10 added to lysozyme (Sigma Cat. L4631) at 2 mg/ml to form a protein marker. Figure 13 shows the same mixture of dyes as described in Figure 10 added to myoglobin protein (Sigma, Cat. M0630) to form a protein marker.
Figure 14 shows how a protein conjugate runs on a 4-20% SDS-polyacrylamide gel (Tris-Glycine buffer) when Remazol Brilliant Blue R (Sigma, Cat. R8001) at 100 mg/ml is mixed with Methyl Vinyl Sulfone (Aldrich Cat. 247197, 5 mg/ml) in various amounts and then added to 10 μl of MBP truncated beta-gal (2 mg/ml) in Lane 2-7, 10 μl of MBP (2mg/ml) in lanes 8-13 and the mixture are incubated at 550C for 4 hours. In order to recognize the protein band on Lanes 6, 7, 12 and 13, Coomassie Blue R-250 (Bio- Rad Cat. No. 161-0400) is needed to stain the gel : Lane 1 : NEB pre-stained marker (NEB, Cat P7708, New
England Biolabs, Inc., Ipswich, MA).
Lane 2: 5μl of Remazol Brilliant Blue R (Sigma, Cat. R8001). Lane 3: 3.75 μl of Remazol Brilliant Blue R, 1.25 μl of Methyl Vinyl Sulfone. Lane 4: 2.5 μl of Remazol Brilliant Blue R, 2.5 μl of Methyl
Vinyl Sulfone.
Lane 5: Remazol Brilliant Blue R (1.25μl), 3.75 μl of Methyl Vinyl Sulfone.
Lane 6: 5 μl of Methyl Vinyl Sulfone. Lane 7: MBP-truncated beta-gal only.
Lane 8: 5 μl of Remazol Brilliant Blue R. Lane 9: 3.75 μl of Remazol Brilliant Blue R, 1.25 μl of Methyl Vinyl Sulfone.
Lane 10: 2.5 μl of Remazol Brilliant Blue R, 2.5 μi of Methyl Vinyl Sulfone. Lane 11 : 1.25 μl of Remazol Brilliant Blue R, 3.75 μl of Methyl Vinyl Sulfone.
Lane 12: 5 μl of Methyl Vinyl Sulfone (5μl).
Lane 13 : MBP only.
Figure 15 shows mixtures as described in Figure 14 conjugated to the same proteins as in Figure 14 except that the Methyl Vinyl Sulfone is substituted by Sulfo-NHS-LC-Biotin (Pierce Biotechnology, Inc. Cat. 21335) at 5 mg/ml.
Figures 16-1 (A, B, C, D, E, F) - Figures 16-2 (G, H, I) show the chemical structures of two Remazol dyes (A and B); Methyl Vinyl Sulfone and Sulfo-NHS-LC-Biotin (C and D); two isothiocyanates, TRITC and FITC (E and F), which are capable of reacting with a primary or a secondary amino group on protein; and l-Chloro-2,4-dinitrobenzene, Monochloro-s-triazine, Tris (2- carboxyethyl) phosphine hydrochloride (G, H and I), which react with an amino group through a chlorine ion.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The problem of forming a protein ladder having bands spaced at predetermined intervals has been solved here by a novel approach in which a protein is covalently bound to one or more dyes and/or optionally one or more non-dye protein-binding molecules. Marker ladders can be readily assembled as desired using a single protein or different proteins and a series of combinations of dyes and/or non-dye protein-binding molecules. Two or more dyes mixed at various ratios are covalently bound to the protein sample to form a sharp band on a gel having a size that differs from the non-dyed protein. When different sized proteins are treated thus, a precise ladder can be constructed (see Figure 1). For example, a pre-stained protein band with a size of 50 Kd can be made from an unstained protein having a size of 37 Kd and two Remazol dyes mixed together in a predetermined ratio such as Remazol Brilliant Blue R at 31% and Remazol Brilliant Yellow at 69% of the mixture to the protein). Without wishing to be limited by theory, it is proposed here that the dye mixture competitively binds to the reactive amino ligand on the protein. Changing the concentration of dyes in a mixture determines the outcome of the competitive binding reaction. The ratio of dyes bound to the protein affects the size of the protein band in a predictable manner and also its color. The colors may span the entire spectrum of light according to the ratio of different dyes used. Examples of the wide range of colors that might be employed in a protein ladder are provided in the Figures. Dyes that produce a fluorescent signal detectable under ultraviolet light may be used in the mixture. Other dyes may be detectable using X-rays.
The use of mixtures of dyes as described herein provides a means to make a desired colored protein by staining the protein with different dyes such as Remazol dyes at selected ratios (see Figures 2-13). For example, to make a green colored protein, the protein can be stained with a Remazol Brilliant Blue R and a Remazol Brilliant Yellow at a ratio of 1: 1. The type of dyes and their combinations for any particular protein are selected to avoid fuzzy bands. For example in Figure 2, the MBP protein marker in Lane 9 is relatively fuzzy compared to the tight band in Lane 8. The sharpest bands occur in lanes 5-8 where lanes 5-6 are a single dye while lanes 7-8 are a mixture of 2 dyes. In Figures 3-6, a series of rainbows of different dye mixes added to a single protein are shown for MBP truncated beta gal, MBP, E.coli RNase III and Myoglobin. Th e observed sharpness of a substantial number of protein bands subjected to different dye mixtures was unexpected.
The position of the protein bands can be modified not only by mixing two or more dyes but also by adding to the mixture or to one dye and a non-dye protein-binding molecule that bind to the protein competitively. The non-dye protein-binding molecule can include a biological macromolecule such as a modified protein or a chemical reagent.
Where a non-dye protein-binding molecule is used, the ladder may be a single color. For example, the protein can be stained with a Remazol reactive dye (e.g. Remazol Brilliant Blue R) plus Methyl Vinyl Sulfone, which is a chemical reagent having a similar reactive group to the Remazol dyes but without the color-inducing aromatic rings (see Figure 14).
In certain circumstances, it is desirable to visualize a protein ladder both in natural light and on an X-ray film. This may be useful in Western Blotting for example. This can be achieved, for example, by staining a protein with varying ratios of a Remazol dye and Sulfo-NHS-LC Biotin (see Figure 15) providing a suitable color and size on the gel. Furthermore, a Remazol dye and a fluorescent reagent (e.g. FITC or TRITC) can be used to stain a protein at a ratio suitable for generating a particular size band on a gel and for visualizing this protein both in natural light and under ultraviolet light.
An advantage of having a protein ladder formed from proteins of different sizes having different colors is that it is immediately possible to conclude how well the gel is running and how much it has already run by noting the position of a band of known size corresponding to a particular color.
All references cited herein as well as Provisional Application No. 60/691,299, are herein incorporated by reference.
EXAMPLES
Sources of reagents used herein are as follows:
Figure imgf000017_0001
Figure imgf000018_0001
Example 1: Preparation of a third-colored protein by staining with two different Remazol dyes at a certain ratio and preparation of a green-colored protein band from staining with blue and yellow dyes.
Preparation of dves: 100 mg of Remazol Brilliant Blue R (Sigma, Cat. R8001) and 100 mg of Remazol Brilliant Yellow GR (Aldrich, Cat. R318914) were weighed and separately dissolved in 1 ml of DH2O to make a Blue dye solution (100 mg/ml) and a Yellow dye solution (100 mg/ml). A purified MBP (MW: 40 Kd, 2 mg/ml in 60 mM Carborate buffer, pH 9.6) was pretreated by adding SDS to 0.2% and TCEP (Sigma, Cat. C4706) to 10 mM and incubating at 950C for 3 min.
Protein staining: A mixture including 500 μl of N- Ethylmorpholine (Aldrich, Cat. 109932, Sigma-Aldrich, St. Louis, MO), 1 ml of a pretreated MBP, 200 μl of Blue dye solution and 300 μl of Yellow dye solution was incubated at 55° C for 4 hours or 37° C for overnight (see Lane 7 in Figure 2). To terminate this reaction, L- Lysine (Sigma, Cat. L5501, Sigma-Aldrich, St. Louis, MO) was added to 10 mM in the dye mixture. A 2 μl aliquot of reaction mixture was analyzed on a Novex 4-20% Tris-glycine gel (Invitrogen, Cat. EC60255, Carlsbad, CA). To remove the free dyes, the reaction mixture was dialyzed in a buffer (10 mM Tris-HCI pH 7.0, 50 mM NaCI) until all excess dyes were removed. The colored protein was stored in a -20° C freezer.
Example 2: Preparation of two light violet-colored proteins by staining with a mixture of two Remazol dyes at a pre-selected ratio.
Preparation of dyes:
100 mg of Remazol Brilliant Violet 5R (Sigma, Cat. R6006) and 100 mg of Remazol Brilliant Yellow GR (Aldrich, Cat. R318914) were weighed and separately dissolved in 1 ml of DH2O to make a Violet dye solution (100 mg/ml) and a Yellow dye solution (100 mg/ml). A purified MBP (40 kd, 2 mg/ml in 6OmM Carborate buffer, pH 9.6) was pretreated by adding SDS to 0.2% and TCEP (Sigma, Cat. C4706) to 10 mM. The preparation was incubated at 95° C for 3 min. For making prestained MBP of size 70Kd, a mixture including 500 μl of 100%N-Ethylmorpholine (Aldrich, Cat. 109932), 1 ml of a pretreated MBP, 400 μl of Violet dye solution and 100 μl of Yellow dye solution (the ratio of violet dye: yellow dye is 4:1) were incubated at 55 0C for 4 hours or 37° C for overnight. For making a prestained MBP having a size of 50Kd, the ratio of Violet dye: Yellow dye used was 2:3 instead of 4: 1 using the same conditions as described above. To terminate the reaction, L-Lysine was added (Sigma, Cat. L5501) to 10 mM in this dye mixture. An aliquot of 2 μl of reaction mixture was tested on a Novex 4-20 % Tris-glycine gel (Invitrogen, Cat. EC60255) to see if reaction worked. To remove the free dyes, the reaction mixture was dialyzed against a buffer (10 mM Tris-HCI pH 7.0, 50 mM NaCI) until all excess dyes were removed. The colored protein was stored in a -20° C freezer.

Claims

What is claimed is:
1. A protein marker, comprising: a protein of predetermined molecular weight covalently bound to (i) two or more dyes; (ii) one or more dyes in a mixture with one or more non-dye protein- binding molecules, or (iii) two or more non-dye protein-binding molecules; such that the protein marker of (i) or (ii) has a distinctive color, and wherein the protein marker of (i), (ii) or (iii) differs from the protein by at least one of molecular weight or pi.
2. A protein marker according to claim 1, wherein the one or more dyes in (i) or (ii) has a sulfone group.
3. A protein marker according to claim 2, wherein the one or more dyes is a Remazol.
4. A protein marker according to claim 1, wherein the one or more dyes in (i) or (ii) has a reactive chlorine.
5. A protein marker according to claim 1, wherein the one or more dyes in (i) or (ii) is an isothiocyanate.
6. A protein marker according to claim 1, wherein the non-dye protein-binding molecule has a sulfone group.
7. A protein marker according to claim 1, wherein the non-dye protein-binding molecule is Sulfo-NHS-LC-Biotin.
8. A protein marker according to claim 1, having a ratio of dyes that are competitively bound to the protein in (i), a ratio of dyes and non-dye protein-binding molecule that are competitively bound to the protein in (ii), or a ratio of non-dye protein-binding molecules that are competitively bound to the protein in (iii), so as to impart an altered molecular weight to the protein when compared with the protein in the absence of any dye or non-dye protein-binding molecule.
9. A protein marker according to claim 1, wherein the altered molecular weight for (i) is greater than the molecular weight of the protein bound to a first of the two or more dyes and less then the molecular weight of the protein bound to a second dye of the two or more dyes.
10. A protein marker according to claim 1, wherein the altered molecular weight for (ii) is greater than the molecular weight of the protein bound to a first dye of the one or more dyes.
11. A protein marker according to claim 1, wherein the altered molecular weight for (iii) is greater than the molecular weight of the protein bound to a first non-dye protein-binding molecule and less then the molecular weight of the protein bound to a second non-dye protein-binding molecule in the mixture of non- dye protein-binding molecules.
12. A protein marker according to claim 1, wherein the protein marker is a member of a set of protein markers in a ladder wherein each member of the set comprises a protein with a distinct size different from other members of the set and the set of markers has at least one color.
13. A protein marker according to claim 1, wherein the protein marker is a member of a set of protein markers in a ladder wherein each member of the set comprises a protein with a distinct size different from other members of the set and the set of markers has two or more colors.
14. A protein marker according to claim 1, wherein the protein marker is one of a set of protein markers each member of the set having a distinct size and color such that at least 3 different colored markers are contained within the set.
15. A protein marker according to claim 1, wherein the two or more dyes in (i) or (ii) are selected so that the dyes competitively bind the protein to generate a protein having a desired color that is different from the color of the dyes before binding to the protein.
16. A method of making a protein marker having a desired size, comprising: (a) preparing a mixture of dyes or a mixture of at least one dye and a second non-dye protein-binding molecule;
(b) adding the dye mixture to a protein having a pre-selected molecular weight for competitive binding of the dye mixture to the protein to form the protein marker; (c) analyzing the protein marker on a gel; and
(d) selecting the mixture forming the protein marker having the desired size.
17. A method of making a ladder from proteins having altered molecular weights or pi; comprising (a) selecting one or more amino group-binding chemical reagents; and
(b) mixing the one or more chemical reagents with a protein for binding to the protein such that the bound protein forms a protein marker in a size ladder or isoelectronic fusing marker ladder capable of being visualized on a gel.
18. A method according to claim 17, wherein the chemical reagent is at least one of Methyl Vinyl Sulfone and Sulfo-NHS-LC- Biotin.
19. A ladder, comprising : a plurality of protein markers according to claim 1, wherein each protein marker has a molecular weight that is different from each of the other protein markers in the ladder.
20. A ladder according to claim 19, wherein the chemical reagent is at least one of Methyl Vinyl Sulfone and Sulfo-NHS-LC- Biotin.
21. A detection means for detecting an assay endpoint, comprising : a first protein linked to a first dye having a first color and a second protein linked to a second dye having a second color such that when the first protein becomes linked to the second protein, the first dye and the second dye produce a third color.
22. A detection means according to claim 21, wherein the linkage of the first protein to the second protein is achieved by intein-mediated ligation.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2069386A2 (en) * 2006-07-21 2009-06-17 Life Technologies Corporation Sharply resolving labeled protein molecular weight standards
GB2512664A (en) * 2013-04-05 2014-10-08 Generon Ltd Sample loading buffer for visualisation of target molecules in polyacrylamide gel electrophoresis
WO2015025054A1 (en) 2013-08-22 2015-02-26 Medizinische Universität Wien Dye-specific antibodies for prestained molecular weight markers and methods producing the same
WO2016087514A1 (en) 2014-12-02 2016-06-09 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Anti-mutant calreticulin antibodies and their use in the diagnosis and therapy of myeloid malignancies

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0963435B1 (en) 1997-01-08 2008-05-28 Invitrogen Corporation Methods for production of proteins

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003070967A2 (en) * 2002-02-20 2003-08-28 Invitrogen Corporation Highly homogeneous molecular markers for electrophoresis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003070967A2 (en) * 2002-02-20 2003-08-28 Invitrogen Corporation Highly homogeneous molecular markers for electrophoresis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COMPTON MARK M; LAPP STACEY A; PEDEMONTE RON: "Generation of multicolored, prestained molecular weight markers for gel electrophoresis" ELECTROPHORESIS, vol. 23, no. 19, September 2002 (2002-09), pages 3262-3265, XP002403202 *
COTTON ET AL: "GENERATION OF A DUAL-LABELED FLUORESCENCE BIOSENSOR FOR CRK-II PHOSPHORYLATION USING SOLID-PHASE EXPRESSED PROTEIN LIGATION" CHEMISTRY AND BIOLOGY, CURRENT BIOLOGY, LONDON, GB, vol. 7, no. 4, April 2000 (2000-04), pages 253-261, XP000989902 ISSN: 1074-5521 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2069386A2 (en) * 2006-07-21 2009-06-17 Life Technologies Corporation Sharply resolving labeled protein molecular weight standards
EP2069386A4 (en) * 2006-07-21 2009-10-28 Life Technologies Corp Sharply resolving labeled protein molecular weight standards
US9733212B2 (en) 2006-07-21 2017-08-15 Life Technologies Corporation Sharply resolving labeled protein molecular weight standards
US10302591B2 (en) 2006-07-21 2019-05-28 Life Technologies Corporation Sharply resolving labeled protein molecular weight standards
GB2512664A (en) * 2013-04-05 2014-10-08 Generon Ltd Sample loading buffer for visualisation of target molecules in polyacrylamide gel electrophoresis
GB2512664B (en) * 2013-04-05 2017-09-13 Daniel Brownleader Michael Sample loading buffer for visualisation of target molecules in polyacrylamide gel electrophoresis
WO2015025054A1 (en) 2013-08-22 2015-02-26 Medizinische Universität Wien Dye-specific antibodies for prestained molecular weight markers and methods producing the same
WO2016087514A1 (en) 2014-12-02 2016-06-09 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Anti-mutant calreticulin antibodies and their use in the diagnosis and therapy of myeloid malignancies

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