1. Take a small tube (1ml / tube) of hydration loading buffer (I) (without DTT, without Bio-Lyte) frozen at -20 ° C from the refrigerator and dissolve it at room temperature.

2. Add 0.01g DTT to the small tube, 2.5ml each of Bio-Lyte 4-6, 5-7, and mix well.

3. Remove 400ml of hydrated loading buffer from the small tube, add 100ml of sample, and mix well.

4. Take the IPG prefabricated strips (17cm pH4-7) frozen and stored at -20 ° C from the refrigerator, and leave them at room temperature for 10 minutes.

5. Linearly add the sample along the edge of the groove in the focusing disk or hydration disk to the left and right. Do not add samples about 1 cm at each end of the tank. The sample solution in the middle must be coherent. Note: Do not generate bubbles. Otherwise it will affect the distribution of protein in the rubber strip.

6. After all the protein samples have been added to the focusing tray or hydration tray, use tweezers to gently remove the protective layer on the prefabricated IPG strip.

7. Distinguish the positive and negative poles of the rubber strip, and gently place the IPG rubber strip face down on the sample solution in the focusing disc or hydration disc so that the positive pole of the strip (marked with +) corresponds to the positive pole of the focusing disc . Make sure that the strip is in close contact with the electrode. Do not get the sample solution on the plastic support membrane on the back of the strip, because these solutions will not be absorbed by the strip. Also be careful not to bubble the solution under the strip. If air bubbles have been generated, use tweezers to gently lift one end of the strip and move the strip up and down until the bubbles are pushed out of the strip.

8. Cover each rubber strip with 2-3ml of mineral oil to prevent the evaporation of liquid during the hydration process of the rubber strip. It is necessary to slowly add mineral oil and slowly add the mineral oil drop by drop on the plastic support film along the strip.

9. Align the positive and negative poles and close the lid. Set the isoelectric focusing program.

10. Focus on the finished strip. Immediately perform equilibration and second-direction SDS-PAGE electrophoresis, otherwise place the strip in the sample hydration tray and store in the refrigerator at -20 ℃.

(2) Second-dimension SDS-PAGE electrophoresis

1. Prepare two pieces of 10% acrylamide gel. With 80ml gel solution, each gel 40ml, the solution was injected into the glass plate sandwich, leaving a space of 1cm at the top, with MilliQ water, ethanol or water saturated n-butanol cover to keep the glue surface flat. Polymerize for 30 minutes. Generally, after the gel is layered with the liquid above, it indicates that the gel has basically polymerized.

2. After the gel is solidified, pour off MilliQ water, ethanol or water-saturated n-butanol on the surface of the separation gel and rinse with MilliQ water.

3. The strips taken out from the -20 ℃ refrigerator are allowed to stand at room temperature for 10 minutes to dissolve.

4. Prepare the gel strip equilibration buffer I.

5. Put the dry thick filter paper on the table first, and put the focused rubber strip on the dry thick filter paper with the rubber side up. Wet another thick filter paper with MilliQ water, squeeze out excess water, and then put it directly on the strip, gently absorb the mineral oil and excess sample on the strip. This can reduce the vertical streaks that appear when staining the gel.

6. Transfer the adhesive strips to the swelling plate, one adhesive strip per tank, and add 5ml of adhesive strip balancing buffer I to the tank with adhesive strips. Place the sample hydration tray on a horizontal shaker and shake slowly for 15 minutes.

7. Prepare the gel strip equilibration buffer II.

8. After the first equilibration is complete, drain or aspirate the strip equilibration buffer I in the sample hydration tray. And use the filter paper to absorb the excess balancing liquid (the rubber strip is erected on the filter paper to avoid loss of protein or damage to the gel surface). Add Strip equilibration buffer II and continue to shake slowly on a horizontal shaker for 15 minutes.

9. Absorb excess liquid between the glass plates above the SDS-PAGE polyacrylamide gel with filter paper. Put the processed second-direction gel on the table, with the long glass plate down and the short glass plate up, and the top of the gel slaughtered about? Br>

10. Heat and dissolve the agarose sealant.

11. Dilute 10 × electrophoresis buffer with a measuring cylinder 10 times to make 1 × electrophoresis buffer. Remove air bubbles from the buffer surface.

12. After the second equilibration is complete, completely drain or aspirate the strip equilibration buffer II in the sample hydration tray. And use the filter paper to absorb the excess balancing liquid (the rubber strip is erected on the filter paper to avoid loss of protein or damage to the gel surface).

13. Remove the IPG strip from the sample hydration tray, grip one end of the strip with tweezers so that the gel surface is completely immersed in 1 × electrophoresis buffer. Then place the adhesive strip with the adhesive side up on the long glass plate of the gel. The rest of the rubber strips operate in the same way.

14. Transfer the SDS-PAGE gel with the glue strip to the glue filling rack, with the short glass plate facing you. Add the low melting point agarose sealant above the gel.

15. Using tweezers, tongue depressor or flat-tipped needle, gently push the strip down to make it fully contact with the polyacrylamide gel surface. Be careful not to create any air bubbles under the strip. When using tweezers, tongue depressor or flat-tipped needle to push the adhesive strip, be careful to push the support film on the back of the gel, and do not touch the adhesive surface.

16. Leave for 5 minutes to allow the low melting point agarose sealant to solidify completely.

17. After the low-melting point agarose sealant is completely solidified. Transfer the gel to the electrophoresis tank.

18. After adding the electrophoresis buffer to the electrophoresis tank, turn on the power, and use the low current (5mA / gel / 17cm) or low voltage at the beginning. After the sample is completely out of the IPG strip and concentrated into a line, add Large current (or voltage) (20-30mA / gel / 17cm), stop electrophoresis when the bromophenol blue indicator reaches the bottom edge.

19. After the electrophoresis is completed, gently pry off the two layers of glass, remove the gel, and cut the corner for marking (wear gloves to prevent contamination of the glue surface).

20. Perform staining.

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