DE4127546C2 - - Google Patents

Description

The invention relates to the method specified in claims 1 to 9 for carrying out a protein separation by SDS electrophoresis, as well as the suitable homogeneous electrophoresis plates according to the Claims 10 to 13 and their use according to Claim 14.

SDS electrophoresis, in which the detergent Sodium dodecyl sulfate (SDS) is used is a relative easy to do and extremely informative Method of separating proteins according to their Molecular mass.

There are already several SDS electrophoresis methods known. From the company Pharmacia LKB, for example SDS gradient finished gels and electrode strips Polyacrylamide with a tris-acetate-tricinate buffer system (ExcelGele) for performing an SDS electrophoresis  brought on the market. The distributed by this company SDS ready gels consist of a collective gel (with a Total polyacrylamide concentration of 5 g / dl and one Degree of crosslinking of 0.03) and a separating gel (with a Total polyacrylamide concentration over a Range continuously changes from 8 to 18 g / dl, and one Degree of crosslinking of 0.03).

However, these gradient 8-18 polyacrylamide SDS gels have the disadvantage that their preparation requires great care and time, so that these products are expensive, and they also dry irregularly, with the result that the gel , deformed together with the carrier or the supporting fabric to which it is applied. In addition, the offered tris-acetate-tricinate buffer system, which contains the Excel gels, has a low buffer capacity because its functional pH value of 7.1 is far from the pK _c values of the trision (8.2), the acetic acid ( 4.6) and tricine (8.0).

The object of the invention was therefore SDS electrophoresis to develop further by using homogeneous gels that are easier and cheaper to manufacture, and one Buffer systems with a higher buffer capacity.

It has now been found that this object can be achieved according to the invention by performing the SDS electrophoresis using homogeneous bulk and separating gels with a specific polyacrylamide content within the range from 4 to 24 g / dl and with a degree of crosslinking of 0 , 02 to 0.05, preferably 0.04, in combination with a tris formate taurinate buffer system, the functional pH of which is in the range from 8.0 to 9.0, preferably at 8.5, and is therefore essential is closer to the pK _c values of the trision (8.2) and the taurine (8.9), so that it has a significantly higher buffer capacity.

The new method for performing the SDS described here Electrophoresis using homogeneous gels in a novel tris formate taurinate buffer system, avoids the labor-intensive and expensive Gradient concentration of the known SDS ready gels and improves the separation of proteins. Since the homogeneous gels used according to the invention easily and are technically easy to manufacture, thereby cheaper very significantly the SDS electrophoresis process.

The concentration of the polyacrylamide (a polymer made from Acrylamide and bisacrylamide) in the invention homogeneous bulk and separating gels used is 4 to 5, preferably 5, g / dl, or 8 to 24, preferably 12, g / dl, and its degree of crosslinking is 0.02 to 0.05, preferably at 0.04.

To increase the low capacity of the known ExcelGel buffer system, a tris-formate-taurinate buffer system was developed in which the formation (the ion of formic acid) as the leading ion, the taurination (the ion of taurine) as the secondary ion, and the trision as the counter ion act. The functional pH of this buffer system is 8.0 to 9.0, preferably 8.5, and thus close to the pK _c value of the trision (8.2) and the taurine (8.9).

Instead of formic acid, a strong acid can also be used (e.g. hydrochloric acid) can be used. Instead of Taurine can also use taurine derivatives and others structure-like connections (e.g. Aminomethanesulfonic acid, 3-aminopropanoic acid, alanine, β-alanine, 2-aminoethanephosphonic acid, 2-aminoethylsulfuric acid, Cysteine, serine and threonine) can be used.

The invention relates to a method for Protein separation by SDS electrophoresis,  which is characterized in that an aqueous solution of protein mixture to be separated on a homogeneous gel layer an electrophoresis plate consisting of a homogeneous Combination gel layer and a homogeneous separating gel layer, in combination with a tris formate taurinate buffer system a carrier or support fabric, applied and after the Put on two paper electrode strips by applying a direct current in a manner known per se an SDS Electrophoresis is performed.

Another object of the invention is a homogeneous Electrophoresis plate for protein separation by SDS electrophoresis using the above described method, consisting of a carrier or a Supporting fabric with a stacking gel layer applied to it and a separating gel layer in combination with a tris Formate taurinate buffer system and one each wear the paper electrode strip.

Preferably, an electrophoresis plate is used, the Collective gel layer polyacrylamide with a degree of crosslinking C = 0.04 in a homogeneous concentration T = 5 g / dl and 30 g / dl glycerin in combination with a tris formate taurinate buffer system contains. Your release gel layer preferably contains Polyacrylamide with a degree of crosslinking C = 0.04 in one homogeneous concentration T = 12 g / dl and 10 g / dl glycerol in a tris formate taurinate buffer system.

An approximately 0.2 mm thick film made of polyester or a net made of polyester or glass fibers used in the (the) that of the gel layer facing surface is hydrophilic.

The buffer system of the stacking gel layer and the The separating gel layer consists of 0.1 to 0.6 mol / l (1.21 up to 7.27 g / dl), preferably 0.389 mol / l (4.71 g / dl), tris (hydroxymethyl) aminomethane (TRIS), 0.1 to 0.4 mol / l (0.38  up to 1.52 ml / dl), preferably 0.3 mol / l (1.14 ml / dl), Formic acid and 0.001 to 0.004 mol / l (0.03 to 0.12 g / dl), preferably 0.002 mol / l (0.06 g / dl), sodium dodecyl sulfate (SDS) and each has a pH of 7.0 to 8.5, preferably from 7.8.

The paper electrode strips contain a buffer system 0.2 to 1.2 mol / l (2.42 to 12.11 g / dl), preferably 0.589 mol / l (7.13 g / dl), TRIS, 0.2 to 1.6 mol / l (2.50 to 15.02 g / dl), preferably 0.802 mol / l (10.04 g / dl), taurine (2-amino-ethanesulfonic acid) and 0.002 to 0.01 mol / l (0.06 to 0.29 g / dl), preferably 0.007 mol / l (0.20 g / dl), SDS with a pH of 8.0 to 9.0, preferably 8.5 (Cathode buffer), or from 0.4 to 1.6 mol / l (4.85 to 19.38 g / dl), preferably 1.295 mol / l (15.69 g / dl), TRIS, 0.25 to 1.50 mol / l (0.95 to 5.72 ml / dl), preferably 1.0 mol / l (3.81 ml / dl), formic acid and 0.002 to 0.01 mol / l (0.06 to 0.29 g / dl), preferably 0.007 mol / l (0.20 g / dl), SDS with a pH of 7.0 to 8.5, preferably of 7.8 (Anode buffer).

According to the invention, SDS electrophoresis can be described as horizontal or vertical or also as two-dimensional electrophoresis be performed. It can be analytical or as a blotting Electrophoresis are used, according to the Performing SDS electrophoresis from each other separated proteins fixed in a manner known per se and be stained with a staining solution. The SDS Electrophoresis is carried out at a voltage of 50 to 600 V. performed within 0.5 to 2 hours.

The process according to the invention can be used for example liver, muscle, marker, spleen, peanut, Globulin, snake venom, Escherichia coli, wheat, Rice, onion, bean, lentil, pea, sunflower, Hazelnut proteins in a concentration of preferably  Reliably separate 0.250 to 0.125 mg / ml. The Proteins are used in a form denatured with SDS.

The fixation of the separated proteins after The SDS electrophoresis is carried out 2 to 3 times each 10 to 15 min with 0.5 g / dl glutardialdehyde in 50 ml / dl Ethanol at 60 ° C or room temperature.

The staining to make the proteins visible takes place in known manner using a coloring solution, preferably a solution of Coomassie Brillantblau R 250 or a silver staining solution.

Applying the samples with those to be separated Proteins are made on the bulk gel with the help of a Applicator strips, with sample volumes of 1 to 20 ul can be abandoned. Up to 50 on a gel Samples are separated at once.

The SDS electrophoresis according to the invention allows one fast and effective separation of proteins in the Molecular mass range from 6000 to 450,000 achieved gangs are sharper than with the known SDS Electrophoresis systems and it becomes a significantly higher one Dissolution (formation of more separation zones) from Protein mixtures achieved.

The homogeneous electrophoresis plates according to the invention can be produced in a technically simple manner, for example as follows (see Figs. 1 and 2 of the accompanying drawing):

In a clear plexiglass box ( Fig. 1) glass plates with vertically glued 0.25 to 1.0 mm, preferably 0.5 mm thick spacing strips and gel-fix foils or gel-fix nets (vertically and successively) are placed (next to each other) Glass plate loosely fixed) until the box is completely full. A buffer solution (water) is first poured in with a tube ( Fig. 2) and then a collection gel solution is introduced under the buffer solution. After polymerization of the collecting gel, which is carried out in the usual way using known catalysts such as tetramethylethylenediamine (TMEDA, TEMED) and ammonium peroxydisulfate [(NH₄) ₂S₂O₈], a collecting gel with a very smooth upper limit is obtained. The excess buffer solution (water) is suctioned off, e.g. B. with a water jet pump and then a separation gel solution for the preparation of the separation gel is poured in from above. After the polymerization of the separating gel has been carried out in the manner described above, the gel layers are removed together with the Gel-Fix films or with the Gel-Fix nets, and each gel layer can be welded into plastic film using a welding device. The homogeneous gels obtained can be stored for at least 12 months if stored at room temperature.

The paper electrode strips are the same Thickness of 6 mm, but different lengths and widths, e.g. B. 260 mm × 12.5 mm. The cathode paper electrode strips contain cathode buffers and the Anode paper electrode strips contain anode buffers (so.). When stored at room temperature, they are Paper electrode strips can be kept for at least 12 months.

The invention is illustrated in the following examples, in which recipes for homogeneous SDS ready gels with different Concentration of the separating gel (T, g / dl) are given.

In Examples 1 to 7 the gel layers have different dimensions, the matrix is made of polyacrylamide, the stacking gel layer (T = 5 g / dl, C = 0.04) makes 1/4 to 1/3 of the entire gel layer, the separating gel layer (T = 8, 10, 12, 14, 16, 18 or 20 g / dl, C = 0.04) makes 3/4 to 2/3 the entire gel layer. The carrier fabric of the SDS  The finished gel is a polyester film (0.2 mm) or a Polyester network (80 µm). The gel layers can at Be stored at room temperature and their shelf life is at least 12 months. Otherwise the buffers in the Collective gel layer and each in the separating gel layer The following table shows the composition (Tab. I):

Table I

Composition of the gel layer buffer

The cathode and anode buffers of the paper electrode strips each have those in Tables II and III below specified compositions:

Table II

Composition of the cathode buffer

Table III

Composition of the anode buffer

Claims (14)

1. A method for performing a protein separation by SDS electrophoresis, characterized in that an aqueous solution of the protein mixture to be separated onto a homogeneous gel layer of an electrophoresis plate, consisting of a homogeneous separating gel layer and a homogeneous collecting gel layer, in combination with a tris formate taurinate. Buffer system, applied to a support or support fabric and, after the application of two paper electrode strips by applying a direct current, SDS electrophoresis is carried out in a manner known per se. 2. The method according to claim 1, characterized in that an electrophoresis plate is used, the Polyacrylamide stacking gel layer with a degree of crosslinking of 0.02 to 0.05, preferably 0.04, in a homogeneous Concentration of 4 to 5, preferably 5 g / dl, and 30 g / dl glycerin in combination with a tris formate taurinate buffer system contains. 3. The method according to claim 1 or 2, characterized in that an electrophoresis plate is used, the Separating gel layer polyacrylamide with a degree of crosslinking C = 0.02 to 0.05, preferably 0.04 in a homogeneous Concentration T = 8 to 24, preferably 12 g / dl, and 10 g / dl glycerin in combination with a tris formate taurinate buffer system contains. 4. The method according to any one of claims 1 to 3, characterized characterized in that an electrophoresis plate with an approximately 0.2 mm thick carrier film made of polyester or a Carrier network made of polyester or glass fibers is used,  the surface of which faces the gel layer is hydrophilic. 5. The method according to any one of claims 1 to 4, characterized characterized in that the buffer system of the stacking gel layer and the separating gel layer in each case consists of 0.1 to 0.6 mol / l (1.21 to 7.27 g / dl), preferably 0.389 mol / l (4.71 g / dl), Tris (hydroxymethyl) aminomethane (TRIS), 0.1 to 0.4 mol / l (0.38 to 1.52 ml / dl), preferably 0.3 mol / l (1.14 ml / dl), formic acid and 0.001 to 0.004 mol / l (0.03 to 0.12 g / dl), preferably 0.002 mol / l (0.06 g / dl), Sodium dodecyl sulfate (SDS) and a pH of 7.0 each to 8.5, preferably from 7.8, and that the paper electrode strips a Buffer system from 0.2 to 1.2 mol / l (2.42 to 12.11 g / dl), preferably 0.589 mol / l (7.13 g / dl), TRIS, 0.2 to 1.6 mol / l (2.50 to 15.02 g / dl), preferably 0.802 mol / l (10.04 g / dl), taurine and 0.002 to 0.01 mol / l (0.06 to 0.29 g / dl), preferably 0.007 mol / l (0.20 g / dl), SDS with a pH from 8.0 to 9.0, preferably from 8.5 (cathode buffer), or from 0.4 to 1.6 mol / l (4.85 to 19.38 g / dl), preferably 1.295 mol / l (15.69 g / dl), TRIS, 0.25 to 1.50 mol / l (0.95 up to 5.72 ml / dl), preferably 1.0 mol / l (3.81 ml / dl), Formic acid and 0.002 to 0.01 mol / l (0.06 to 0.29 g / dl), preferably 0.007 mol / l (0.20 g / dl), SDS with a pH from 7.0 to 8.5, preferably from 7.8 (anode buffer) contain. 6. The method according to any one of claims 1 to 5, characterized characterized in that the SDS electrophoresis as horizontal or vertical electrophoresis, or as two-dimensional Electrophoresis is performed. 7. The method according to any one of claims 1 to 6, characterized characterized in that the SDS electrophoresis in a  Voltage from 50 to 600 V within 0.5 to 2 hours is carried out. 8. The method according to any one of claims 1 to 7, characterized characterized in that SDS electrophoresis as analytical or blotting electrophoresis is performed. 9. The method according to any one of claims 1 to 8, characterized characterized in that after performing the SDS electrophoresis the separated proteins 2 to 3 times each 10 to 15 min with 0.5 g / dl glutardialdehyde in 50 ml / dl ethanol Fixed at 60 ° C or room temperature and then by means of a Staining solution to be stained. 10. Homogeneous electrophoresis plate for performing a Protein separation by SDS electrophoresis, especially after the method according to any one of claims 1 to 9, characterized characterized in that it consists of a carrier or support fabric and a homogeneous stacking gel layer applied thereon and one homogeneous separating gel layer in combination with a tris Formate taurinate buffer system, and one each wear the paper electrode strip. 11. Electrophoresis plate according to claim 10, characterized characterized in that the collecting gel layer with polyacrylamide a degree of crosslinking C = 0.02 to 0.05, preferably 0.04, in a homogeneous concentration T = 4 to 5, preferably 5 g / dl and 30 g / dl glycerin in combination with a tris formate Contains taurinate buffer system. 12. Electrophoresis plate according to claim 10 or 11, characterized characterized in that the separating gel layer with polyacrylamide a degree of crosslinking C = 0.02 to 0.05, preferably 0.04 g / dl in a homogeneous concentration T = 8 to 20, preferably 12 g / dl and 10 g / dl glycerin in combination with a tris Contains formate taurinate buffer system.   13. electrophoresis plate according to one of claims 10 to 12, characterized in that the buffer system of The bulk gel layer and the separating gel layer each consist of 0.1 to 0.6 mol / l (1.21 to 7.27 g / dl), preferably 0.389 mol / l (4.71 g / dl) TRIS, 0.1 to 0.4 mol / l (0.38 to 1.52 ml / dl), preferably 0.3 mol / l (1.14 ml / dl) formic acid and 0.001 to 0.004 mol / l (0.03 to 0.12 g / dl), preferably 0.002 mol / l (0.06 g / dl) SDS and a pH value of 7.0 to 8.5, preferably from 7.8, and that the paper electrode strips each a buffer system from 0.2 to 1.2 mol / l (2.42 to 12.11 g / dl), preferably 0.589 mol / l (7.13 g / dl) TRIS, 0.2 to 1.6 mol / l (2.50 to 15.02 g / dl), preferably 0.802 mol / l (10.04 g / dl) taurine and 0.002 to 0.01 mol / l (0.06 to 0.29 g / dl), preferably 0.007 mol / l (0.20 g / dl) SDS with a pH of 8.0 to 9.0, preferably 8.5 (Cathode buffer), or from 0.4 to 1.6 mol / l (4.85 to 19.38 g / dl), preferably 1.295 mol / l (15.69 g / dl) TRIS, 0.25 to 1.50 mol / l (0.95 to 5.72 ml / dl), preferably 1.0 mol / l (3.81 ml / dl) formic acid and 0.002 to 0.01 mol / l (0.06 to 0.29 g / dl), preferably 0.007 mol / l (0.20 g / dl) SDS with a pH of 7.0 to 8.5, preferably 7.8 (Anode buffer) included. 14. Use of the homogeneous electrophoresis plates after one of claims 10 to 13 for carrying out a Protein separation by SDS electrophoresis.