Discontinuous nondenaturing coagulation limb electrophoresis experiments

Summary

Non-denaturing gel electrophoresis, also known as natural gel electrophoresis. The separation of a protein in a gel depends on its charge and molecular size. While sieving different sized proteins by acrylamide gel pore size, highly charged proteins will swim faster under the acidic pH conditions of the separating gel. This method can distinguish between molecules that differ by only one unit of charge. Compared to SDS-PAGE electrophoresis, nondenaturing gels greatly reduce the chance of protein denaturation occurring. Source: Handbook of Protein Technology

Operation method

Discontinuous nondenaturing condensed limb electrophoresis

Principle

Non-denaturing gel electrophoresis is usually performed under high pH (8.8) buffer conditions. At this point, most proteins are negatively charged and migrate toward the anode.

Materials and Instruments

Protein solutions
Acrylamide Bisacrylamide Tris Alkali Hydrochloric acid Ammonium persulfate TEMED Glycine Glycerol Bromophenol blue Methanol Glacial acetic acid
Bio-Rad Min-Protean II Gel Electrophoresis Tanks Microsyringe or Microsampler Small Containers for Staining and Decolorization

Move

1. Preparation of working solution

2. working solution dosage

(1) Volume of solution for preparing electrophoresis gels of different thicknesses

Preparation of two 6 cm×8 cm gels

The preparation volume should be slightly more than the actual amount.

(2) Calculation of X% separation gel

3. Preparation of Separation Gel

(1) Assemble the mold for gel filling according to the instruction manual;

(2) Mix Liquid A, Liquid B and water in a small triangular flask or test tube (because acrylamide is neurotoxic, gloves should be worn). (Due to the neurotoxicity of acrylamide, gloves should be worn);

(3) Add ammonium persulfate with gentle stirring and TEME mix. Since polymerization occurs in this step, it is important to move quickly;

(4) Pipette the gel solution into the gel mold;

(5) Immediately after adding the separated gel, gently cover the top of the gel with 1 to 5 mm of Mizutaki;

(6) leave 30 ~ 60 min, to be gel polymerization; when the gel polymerization, in the separation between the gel and water layer will appear a clear interface, then the gel will be slightly tilted. Judge whether the gel is polymerized.

4. Preparation of stacking gel

(1) Remove the water layer covering the separator gel;

(2) In a small triangular flask or test tube, mix liquids A and C with distilled water;

(3) Add ammonium persulfate and TEMED with gentle stirring and mix well;

(4) Aspirate the stacking gel solution on top of the separating gel until the solution reaches the top of the front glass plate.

(5) Carefully insert a comb between the two plates until the bottom of the comb teeth reaches the top of the front glass plate, avoiding air bubbles at the end of the comb teeth.

(6) Allow to stand for 30 minutes for the buildup to polymerize;

(7) After the gel has polymerized, carefully remove the comb without cracking the spiking holes;

(8) Put the gel into the electrophoresis tank and connect the positive and negative electrodes;

(9) Add the electrophoresis buffer into the inner and outer electrophoresis tanks. Make sure the top and bottom of the gel are immersed in the buffer.

5. Sample preparation

(1) Sample volume of spiked wells (Mini-Gel system)

(2) Mix the protein sample and sample buffer (20 ul + 5 ul) in an Eppendorf tube;

(3) Add the sample to the spiking wells using a micro syringe;

(4) Before adding the different samples. Rinse the syringe by aspirating a small amount of electrophoresis buffer with the syringe to avoid mutual contamination between samples.

6. Electrophoresis

(1) Connect the electrode plug to the appropriate electrode. For gels of pH 8.8, the current should flow to the anode;

(2) Adjust the voltage to 100 to 200 V;

(3) Migrate the front of the tracer dye to about 1-5 mm from the bottom of the gel;

(4) Turn off the power;

(5) Unplug the electrode;

(6) Remove the electrophoresis gel plate, put it in a safe place, and gently pry off the glass plates on both sides of the gel, so that the gel is stuck on one of the plates.

7. Gel staining

(1) Wearing gloves, transfer the gel to a petri dish containing about 20 ml of Caulmers Brilliant Blue;

(2) Shake the gel slowly on a shaker for 5 to lO min for 0.75 mm and 10 to 20 min for 1.5 mm;

(3) Remove the staining solution;

(4) Add about 50 ml of decolorizing solution and continue to shake gently;

(5) For complete decolorization, it is necessary to change the decolorizing solution several times and shake overnight; the gel can be stored dry.

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