Preparation of protein gel electrophoresis gels

Summary

Protein gel electrophoresis is commonly used to (1) analyze molecular biology, genetics, and biochemistry (2) preparative techniques (3) partially purify molecules prior to detection using certain methods such as mass spectrometry (MS), polymerase chain reaction (PCR), cloning techniques, DNA sequencing, or immunoblotting.

Operation method

Preparation of protein gel electrophoresis gels

Principle

Protein samples are heated with the ionic detergent sodium dodecyl sulfate (SDS) and thioethanol to denature the proteins, and the disulfide bonds within and between the peptide chains are reduced and the peptide chains are opened. The opened peptide chain is negatively charged by hydrophobic interaction and SDS binding, and the peptide chain migrates to the positive pole in the gel under the action of electric field during electrophoresis. Different peptide chains are gradually separated during migration due to different resistance during migration, and their relative mobility is linearly related to the logarithm of molecular weight.

Materials and Instruments

Acrylamide
Deionized water SDS Concentrated hydrochloric acid Tris Amine persulfate TEMED Glycine
Electrophoresis tanks Electrophoresis apparatus Samplers Pipette tips

Move

1. Install the glass plates according to the instructions of the vertical electrophoresis tank, determine the concentration and volume of the separator gel to be prepared, and prepare the separator gel according to the ingredients listed in the "Solution for Preparation of Separator Gel for Tris-Glycine SDS Polyacrylamide Gel Electrophoresis" (see Appendix at the end of the book). 2;

2. Rapidly inject the separation gel into the gap between the two glass plates, leaving the space required for filling the cumulus gel (add 1 cm to the length of the teeth of the comb, and carefully cover a layer of 0.1% SDS (when the concentration of acrylamide is ≤8%), or isobutanol, or water (when the concentration of acrylamide is ≥10%) with a dropper, which prevents the oxygen from diffusing into the gel and inhibits the polymerization reaction. Place the gel vertically at room temperature;

3. after the polymerization of the separator gel is complete, pour out the covering layer liquid and wash the top of the gel several times with deionized water to remove unpolymerized acrylamide and to exclude as much liquid from the gel as possible;

4. Determine the volume of gel concentrate to be prepared, prepare the required gel concentrate according to the "Solution for Preparation of Gel Concentrate for Tris-Glycine SDS Polyacrylamide Gel Electrophoresis" (see the Appendix at the end of the book), and then pour the gel concentrate directly onto the separator gel, immediately insert a clean matching comb to avoid air bubbles, and then add the gel concentrate to fill the gaps between the combs. the space between the combs. After the gel concentrate is polymerized, pull out the comb to form the spiking hole;

5. Dilute the Tris-glycine electrophoresis buffer storage solution 5 times with deionized water, pour it into the electrophoresis tank, and fill the spiking hole with air bubbles.

Caveat

1. Because the surface of silicone rubber and glass plate related to gel polymerization is not smooth and clean, it will cause the gel plate to peel off from the glass plate or silicone rubber strip during electrophoresis, resulting in bubbles or slippery gel; the gel plate is easy to break when peeling off the gel; in order to prevent the phenomenon, the equipment used should be cleaned rigorously. Silicone rubber strip groove, sample tank template and electrophoresis tank with foam sponge dipped in "clean" carefully cleaned. The glass plate is immersed in potassium dichromate wash solution for 3-4h or 0.2mol/L KOH alcohol solution for more than 20min, washed with water, and then foam sponge dipped in "Wash and Clean" repeatedly brushing, and finally rinsed with distilled water, directly dry or rinsed with ethanol and then dried.

2. When installing the electrophoresis tank and the silicone rubber frame with long and short glass plates, the position should be correct, and the fixing screws should be tightened with even force so as to avoid leakage of buffer liquid. The comb teeth of the sample tank plate should be flat and smooth.

3. In the discontinuous electrophoresis system, pre-electrophoresis can only be carried out after the separation of the gel country. Concentrated gel can only be prepared after washing the gel surface. After the preparation of concentrated gel, pre-electrophoresis should not be carried out to make full use of the concentration effect of concentrated gel.

4. When electrophoresis, the positive and negative poles between electrophoresis instrument and electrophoresis tank should not be connected incorrectly, so as to avoid the samples swimming in the opposite direction, and the appropriate current and voltage should be used when electrophoresis, and the effect of electrophoresis will not be affected if it is too high or too low.

5. SDS purity: In SDS-PAGE, high purity SDS is required, and commercially available chemically pure SDS should be recrystallized once or twice before use. Recrystallization method is as follows: weigh 20g SDS in a round-bottomed flask, add 300ml of anhydrous ethanol and about half an ox-horn spoon of activated carbon, connect a condenser tube on the flask, heat it in a water bath until the ethanol is slightly boiling, reflux for about 10min, and filter it through a hot Brinell's funnel while it is still hot. The filtrate should be clear, cooled to room temperature and then transferred to a refrigerator at -20°C overnight. The next day with a pre-cooled Brinell funnel filtration, and then a small amount of -20 ℃ pre-cooled anhydrous ethanol to wash the white precipitate for 3 times, as far as possible to dry, the white crystals will be placed in a vacuum desiccator drying or placed below 40 ℃ in the oven drying.

6. When treating protein samples with SDS, the samples were held in boiling water for 3--5min each time to avoid the presence of substable polymers.

7. It is desirable that the relative mobility of standard proteins be uniformly distributed between 0.2 and 0.

8. It is worth pointing out that every time the molecular weight of the unknown substance is determined, the standard curve should be prepared with the standard protein at the same time, instead of using the standard curve in the past. The molecular weights determined by this method are only the molecular weights of their subunits or single peptide chains, not the complete molecular weights. To obtain an accurate molecular weight range, it is best to correct for other methods of determining the molecular weight of proteins. This method is better for the molecular weight determination of globulin and fibrous proteins, while the molecular weight determination of glycoproteins, collagen, etc. varies greatly.

8. Requirements for samples: Samples with low ionic strength should be adopted. If the ionic strength of the sample is high, it should be dialyzed or desalted by ion exchange. When adding samples, keep the concave adding groove gel surface straight. Addition of 10-15 μl of sample is appropriate, such as the sample is a dilute liquid, in order to ensure that the zone is clear, the amount of sample can be increased, while the sample should be dissolved in the concentration of liquid to increase two times or higher.

9. Because the gel contains SDS, the direct preparation of dry plate will produce cracking phenomenon. If you need to make a dry plate, with 25% isopropyl alcohol containing 7% acetic acid soak, and often change the liquid, until the SDS is removed (about 2-3 days), before the preparation of dry plate. For convenience, a photographic method is often used to preserve the photographs.

Common Problems

Discontinuous polyacrylamide gel electrophoresis contains two different buffer compositions, ph values, and gel pore sizes, and the potential gradients formed during electrophoresis are all different, resulting in a concentration effect, a charge effect, and a molecular sieve effect:

(1) Concentration effect: The sample is concentrated into a highly concentrated thin layer of sample by the concentration gel when electrophoresis is started;

(2) Charge effect: in the separation gel with uniform potential gradient and PH, due to the different isoelectric points of each protein with different charges, different gravitational forces in the electric field, after a period of electrophoresis, a variety of proteins are arranged in a certain order into a protein zone;

(3) The pore size of the separator gel is small, and proteins with different molecular weights and shapes are separated by the separator gel because of the different degree of blockage and the different mobility of the proteins.

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