CN104483294A - Applications of calcium carboxylic acid and derivatives thereof in phosphorylated protein fluorescence detection - Google Patents
Applications of calcium carboxylic acid and derivatives thereof in phosphorylated protein fluorescence detection Download PDFInfo
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Abstract
The present invention relates to a phosphorylated protein fluorescence detection technology, specifically to applications of calcium carboxylic acid and derivatives thereof in phosphorylated protein fluorescence detection. The present invention further provides a method for adopting calcium carboxylic acid to carry out phosphorylated protein fluorescence detection, wherein the method comprises dyeing after protein sample electrophoresis, double-step decolorization, and detection. According to the invention, advantages of good specificity, low detection limit, simple operation, stable reproducibility, good dyeing effect, good linearity, good reversibility, good compatibility, safe and environmental protection use, low cost and the like are provided.
Description
Technical field
The present invention relates to phosphorylating protein detection technique of fluorescence, relate in particular to a kind of new fluorescent dye of phosphorylating protein.
Background technology
In life science field, be unable to do without the base substance protein of life and the research of DNA, also just be unable to do without the detection technique to protein.Especially recent years, along with the develop rapidly of genomics and proteomics, us are made to have had higher requirement to the separation detection technique of protein.
The detection method of protein has a lot, comprises nephelometry, Kjeldahl's method, absorption photometry, fluorimetry, and resonance rayleigh light scattering method, high performance liquid chromatography, capillary electrophoresis analysis method etc. that new development is got up.The detection method that fluorimetry is current most study, most widely used, operation is comparatively easy, sensitivity is higher.
Protein phosphorylation modification is the most common is also one of most important protein post-translational modification mode, also be one of focus of current proteomics research, even there is the deficiency of specificity detection method for current research, Phosphorylation events occurs on the amino acid residues such as tyrosine, threonine, serine substantially.This modification of protein and many vital movements such as propagation, differentiation, growth, apoptosis of cell closely bound up, prove according to years of researches, protein phosphorylation and many clinical diseases have complicated relation.Therefore the breakthrough point that the strong detection method of a kind of specificity becomes research is developed.
On existing gel, phosphorylating protein detection method comprises Pro-Q Diamond detection technique, westernblot detection technique, and the Stains-All detection technique etc. of classics.Wherein Pro-Q Diamond is the detection method that current specificity is the strongest, most widely used, operation is comparatively easy, sensitivity is higher.
Calcium carboxylate and derivant thereof are applied to phosphorylating protein fluoroscopic examination by the present invention first, compared to other detection techniques, have detectability low, and specificity waits many merits by force, will be with a wide range of applications in proteomics research.
Summary of the invention
The object of the invention is to set up a kind of calcium carboxylate and the application of derivant in phosphorylating protein fluoroscopic examination thereof.
Calcium carboxylate related compound of the present invention refers to calcium carboxylate to be the sodium salt, sylvite, hydrochloride, sulfate, hydrosulfate, nitrate, tannate, hydriodate etc. of parent nucleus.
Calcium carboxylate parent nucleus is:
Here is the available Gel Detection Method of the present invention one, and it comprises step:
1) to adding fluorescent dye liquid dyeing 10 ~ 40min in the protein example after SDS-PAGE electrophoresis, wherein said dyeing liquor is containing the calcium carboxylate of molar concentration than 10 ~ 50 μMs, by the 1,2-PD of liquor capacity than 10 ~ 50%, and press liquor capacity than 0.1 ~ 5% acetic acid;
2) the complete gel of dyeing is placed in destainer 1 to decolour 10 ~ 30min, wherein said destainer is containing by the methyl alcohol of liquor capacity than 10 ~ 30%, molar concentration than 50 ~ 200mM sodium acetate, Al
3+concentration molar concentration ratio be 30 ~ 70 μMs;
3) the complete gel of decolouring is placed in destainer 2 decolouring 0.5 ~ 5min, wherein said destainer is containing by the ethanol of liquor capacity than 10 ~ 50%, by the 1,2-PD of liquor capacity than 5 ~ 20%;
4) detect, determined wavelength is 350 ~ 500nm, is preferably 450nm.
The inventive method tool has the following advantages:
1) high sensitivity: fluorescent dye is higher than the sensitivity of common dye doubly a lot, and can match in excellence or beauty classical phosphorylated protein detection kit Pro-Q Diamond;
2) rapidly simple to operate: operation steps is few, can complete in 45min;
3) reappearance is stablized: affect less by external conditions such as temperature, shaking table hunting frequencies;
4) dyeing background is good;
5) linear relationship is good: accurately, can carry out semiquantitative determination in a big way to protein;
6) good reversibility: easily decolour;
7) compatible good: can with mass spectrometer and other colouring method highly compatibles such as MALDI-TOF;
8) use safety, environmental protection, low toxicity;
9) specificity is strong;
10) cost is low.
Accompanying drawing explanation
Fig. 1. the chemical constitution of calcium carboxylate;
Fig. 2. calcium carboxylate fluorescence colour and other conventional decoration method sensitivity and specific comparisons.Wherein (A, D) is calcium carboxylate decoration method, and (B, E) is Pro-Q Diamond decoration method, and (C, F) is SYPRO-Ruby decoration method.The sample that (A, B, C) selects is commercial protein standard substance, be respectively transferrins (transferrin) from top to bottom, bovine serum albumin(BSA) (bovine serum albumin), ovalbumin (OVA), yolk high phospholipid albumen (phosvitin), alpha-casein (α-casein), beta-casein (β-casein).Protein volume containing the sample (every band) is as follows: band 1,500ng; Band 2,250ng; Band 3,125ng; Band 4,64ng; Band 5,32ng; Band 6,16ng; Band 7,8ng; Band 8,4ng; Band 9,2ng; Band 10,1ng.(D, E, F) selects mouse brain total protein as biological actual sample, loading electrophoresis after two-fold dilution.
Fig. 3. calcium carboxylate fluorescence colour specificity is investigated.Adopt different detection method, (A) 1,2,3 be respectively (1) westernblot detection method, (2) calcium carboxylate decoration method, (3) Pro-Q Diamond decoration method, experiment selects mouse brain total protein and rat heart albumen as biological actual sample respectively.(B) calcium carboxylate decoration method is respectively, Pro-Q Diamond decoration method, SYPRO-Ruby decoration method, alpha-casein (α-casein) is selected in experiment, beta-casein (β-casein) albumen as sample, the specificity of further investigation method, protein volume containing the sample (every band) 250ng, "+" representative is through the process of alkaline phosphatase dephosphorylation, and "-" representative is without the process of alkaline phosphatase dephosphorylation.
Fig. 4. calcium carboxylate fluorescence colour and Pro-Q Diamond decoration method linear dynamic range compare.(A) calcium carboxylate decoration method; (B) Pro-Q Diamond decoration method.The standard protein selected is respectively yolk high phospholipid albumen (phosvitin), alpha-casein (α-casein), beta-casein (β-casein).After dyeing, protein belt Tina 2.09 image analysis software analysis.
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be appreciated that these embodiments are only for illustration of the present invention, and can not limit the scope of the invention.
Embodiment 1 calcium carboxylate dyes
Calcium carboxylate phosphorylating protein fluorescent dye experiment adopts following step to carry out:
(1) dye: the gel after washing is positioned over 50ml 30 μMs of calcium carboxylates, 20%1,2-propylene glycol, dye 25 minutes in the dyeing liquor of 0.5% acetic acid.
(2) 1 is decoloured: the complete gel of dyeing is positioned over 50ml 30% ethanol, 100mM sodium acetate, 40 μMs of Al
3+decolour 20 minutes in destainer.
(3) 2 are decoloured: the complete gel of decolouring is positioned over 50ml 30% ethanol, decolours 1 minute in 10%1,2-propylene glycol destainer.
(4) gel be colored is placed directly on transilluminator, the transmission of 450nm place, recording image.
Carry out protein staining with the sodium salt of calcium carboxylate, sylvite, hydrochloride, sulfate, hydrosulfate, nitrate, tannate, hydriodate respectively according to the method described above, result shows the testing result that all can obtain being similar to calcium carboxylate with these derivants.SDS-PAGE carries out with reference to " Molecular Cloning: A Laboratory guide " third edition relevant portion.
Experimental example 1 calcium carboxylate and other dyeing Contrast on effects
Adopt coloured differently method to dye, wherein (A) is calcium carboxylate decoration method, and (B) is Pro-Q Diamond decoration method, and (C) is SYPRO-Ruby dye method.The sample selected is commercial protein standard substance, be respectively transferrins (transferrin) from top to bottom, bovine serum albumin(BSA) (bovine serum albumin), ovalbumin (OVA), yolk high phospholipid albumen (phosvitin), alpha-casein (α-casein), beta-casein (β-casein).Protein volume containing the sample (every band) is as follows: band 1,500ng; Band 2,250ng; Band 3,125ng; Band 4,64ng; Band 5,32ng; Band 6,16ng; Band 7,8ng; Band 8,4ng; Band 9,2ng; Band 10,1ng.Result is as shown in Fig. 2 (A, B, C), and the sensitivity of calcium carboxylate decoration method is consistent with Pro-Q Diamond decoration method, and calcium carboxylate decoration method specificity is consistent with Pro-Q Diamond decoration method.Wherein calcium carboxylate adopts the method for embodiment 1 to carry out, and Pro-Q Diamond and SYPRO-Ruby decoration method are carried out respectively in the following manner:
Pro-Q Diamond
[1]：
(1) fixing: the gel after electrophoresis to be positioned in the immobile liquid of 50ml 50% methyl alcohol/10% acetic acid and to fix 2 times, each 30 minutes (mild jolting);
(2) wash: gel is positioned over mild jolting in 50ml deionized water, washs 3 times, each 10 minutes;
(3) dye: the gel after washing is positioned in 50ml dyeing liquor and dyes 60 ~ 90 minutes;
(4) decolour: the complete gel of dyeing is positioned in 50ml destainer and decolours 3 times, each 30 minutes.
SYPRO-Ruby
[2]：
(1) dye: the gel after electrophoresis is positioned in 50ml SYPRO Ruby and dyes 3 hours;
(2) decolour: the gel that dyeing terminates is positioned in the destainer of 50ml 10% methyl alcohol/7% acetic acid and decolours 30 minutes.
Experimental example 2 calcium carboxylate decoration method practicality is investigated
Select mouse brain total protein as biological actual sample, the practicality of investigation method.In fig. 2, (D) is calcium carboxylate decoration method, and (E) is Pro-Q Diamond decoration method, and (F) is SYPRO-Ruby decoration method.Result shows, and Pro-Q Diamond is as phosphorylating protein detection technique gold criterion, and the protein band that this detection technique detects is very close with the protein band that calcium carboxylate detects, and imply that calcium carboxylate has good practicality and selectivity.
Experimental example 3 calcium carboxylate decoration method specificity is investigated
Adopt different detection method, Fig. 3 (A) 1,2,3 is respectively (1) westernblot detection method, (2) calcium carboxylate decoration method, (3) are Pro-Q Diamond decoration method, experiment selects mouse brain total protein and rat heart albumen as biological actual sample respectively, the protein band that westernblot detection method, Pro-Q Diamond decoration method protein band and calcium carboxylate detect is very close, imply that calcium carboxylate has good specificity.Fig. 3 (B) is respectively calcium carboxylate decoration method, Pro-Q Diamond decoration method, SYPRO-Ruby decoration method, alpha-casein (α-casein) is selected in experiment, beta-casein (β-casein) albumen is as sample, the specificity of further investigation method, protein volume containing the sample (every band) 250ng, "+" representative is through the process of alkaline phosphatase dephosphorylation
[3,4], "-" representative is without the process of alkaline phosphatase dephosphorylation, and result display calcium carboxylate has good specificity.
The contrast of experimental example 4 calcium carboxylate fluorescence colour and Pro-Q Diamond fluorescence colour linear dynamic range
Standard protein (the yolk high phospholipid albumen (phosvitin) of three kinds of different molecular weights, alpha-casein (α-casein), beta-casein (β-casein)) be separated in 10% polyacrylamide gel, through (A) calcium carboxylate decoration method; (B), after the dyeing of Pro-Q Diamond decoration method, with the linearity curve of Tina 2.09 image software analytical standard albumen applied sample amount and band intensity, thus the range of linearity of two kinds of colouring methods is compared.Standard protein applied sample amount scope is yolk high phospholipid albumen (phosvitin) 8 ~ 1000ng, alpha-casein (α-casein) 4 ~ 1000ng, beta-casein (β-casein) 4 ~ 1000ng.Show calcium carboxylate decoration method by Fig. 4, with Pro-Q Diamond decoration method, there is the consistent range of linearity.
List of references:
[1]Orsatti L，Forte E，Tomei L，et al.2-D Differencein gel electrophoresis combined with Pro-Q Diamond staining：A successful approach for the identification of kinase/phosphatase targets.Electrophoresis 2009，30(14)：2469-2476
[2]Kiera N.Berggren，Birte Schulenberg，Mary F.Lopez.，et al.An improved formulation of SYPRO Ruby protein gel stain：Comparison with the original formulation and with a ruthenium II tris(bathophenanthroline disulfonate)formulation.Proteomics 2002，2：486-498
[3]Ksiezak-Reding，H.，Yen，S.H.，J.Neurosci.1987，7，3554-3560.
[4]Nakamura，K.，Masuyama，E.，Wada，S.，Okuno，M.，J.Biochem.Biophys.Methods 1990，21，237-245.
Claims (9)
1. calcium carboxylate and the application of derivant in phosphorylating protein fluoroscopic examination thereof.
2. apply as claimed in claim 1, it is characterized in that described calcium carboxylate derivant comprises the sodium salt, sylvite, hydrochloride, sulfate, hydrosulfate, nitrate, tannate, hydriodate etc. of this carboxylic acid.
3. a phosphorylating protein fluorescence detection method, it comprises step:
1) the protein example gel after SDS-PAGE electrophoresis is placed in fluorescent dye liquid dyeing 10 ~ 40min, wherein said dyeing liquor is containing the calcium carboxylate of molar concentration than 10 ~ 50 μMs, by the 1,2-PD of liquor capacity than 10 ~ 50%, and press liquor capacity than 0.1 ~ 5% acetic acid;
2) the complete gel of dyeing is placed in destainer 1 to decolour 10 ~ 30min, wherein said destainer is containing by the methyl alcohol of liquor capacity than 10 ~ 30%, molar concentration than 50 ~ 200mM sodium acetate, Al
3+concentration molar concentration ratio be 30 ~ 70 μMs;
3) the complete gel of decolouring is placed in destainer 2 decolouring 0.5 ~ 5min, wherein said destainer is containing by the ethanol of liquor capacity than 10 ~ 50%, by the 1,2-PD of liquor capacity than 5 ~ 20%;
4) detect.
4. method as claimed in claim 3, is characterized in that, step 1) in time of dyeing be 25min.
5. the method as described in any one of claim 2 ~ 4, it is characterized in that, step 2) the concentration molar concentration ratio of calcium carboxylate is 30 μMs in dyeing liquor, 1, the concentration of 2-propylene glycol is 20% by liquor capacity ratio, and the concentration of acetic acid is 0.5% by liquor capacity ratio.
6. the method as described in any one of claim 2 ~ 4, is characterized in that, step 2) concentration of ethanol is 30% by liquor capacity ratio in destainer 1, the concentration molar concentration of sodium acetate is than being 100mM, Al
3+concentration molar concentration ratio be 40 μMs.
7. the method as described in any one of claim 2 ~ 4, is characterized in that, step 2) bleaching time is 20min.
8. the method as described in any one of claim 2 ~ 4, is characterized in that, step 3) concentration of ethanol is 30% by liquor capacity ratio in destainer 2, the concentration of 1,2-PD is 10% by liquor capacity ratio.
9. the method as described in any one of claim 2 ~ 4, is characterized in that, step 3) bleaching time is 1min.
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Cited By (3)
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| CN105510289A (en) * | 2015-12-02 | 2016-04-20 | 温州医科大学 | Application of anthracene chrome red A (ACRA) and derivative thereof to phosphorylation protein fluorescence detection |
| CN105510289B (en) * | 2015-12-02 | 2019-01-04 | 温州医科大学 | The application of anthracene chrome red A (ACRA) and its derivative in phosphorylating protein fluorescence detection |
| WO2022001824A1 (en) * | 2020-07-01 | 2022-01-06 | 山东第一医科大学第二附属医院 | Kit and method for detecting pd-l1 gene mutations in circulating tumor cells in peripheral blood of patient with small cell lung cancer |
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