Hydration loading (passive loading)

1. Remove the IPG strip from the refrigerator and let it stand for 10 min at room temperature.

2. Add the sample linearly from left to right along the edge of the hydration groove. The ends of the groove are not added about 1cm each, and the sample liquid in the middle must be continuous. Note: Do not create bubbles, otherwise it will affect the distribution of protein in the strip.

3. Use a pair of tweezers to gently remove the protective layer from the IPG strip. Note: The alkaline end is weak and should be handled with care.

4. Gently place the IPG strip face down on the sample solution in the hydration tray. Note: Do not get the sample solution on the back of the strip because these solutions will not be absorbed by the strip; it will also create bubbles in the solution below the strip. If bubbles are generated, gently lift one end of the strip with tweezers and move the strip up and down until the bubbles are driven away.

5. Place for 30~45min. Most of the sample is absorbed by the strip. Slowly add mineral oil along the strip. Each gel is treated with 3ml (17cmIPG) to prevent evaporation of the liquid during the hydration of the strip.

6. Set the isoelectric focusing instrument to hydrate at -20 °C for 11~15h.

First isoelectric focusing

1. Place the paper electrode on the positive and negative electrodes of the focusing disk and add ddH ₂ O 5~8μl to wet.

2. Remove the hydrated strip, lift one end to drain the mineral oil, face down, and place it on the just wetted filter paper to hybridize to remove insoluble material on the surface.

3. Place the IPG strip face down in the focus tray. The positive side of the strip (marked with +) corresponds to the positive side of the focus disc, ensuring that the strip is in intimate contact with the electrode.

4. Cover each strip with 2 - 3 ml of mineral oil.

5. For positive and negative, cover. Set the isoelectric focusing program.

6. Focus on the finished strip and immediately perform a balanced, second-direction SDS-PAGE electrophoresis. Or put the strip in the sample hydration tray, store in a refrigerator at 20 °C, remove the strip before electrophoresis, and let it dissolve at room temperature for 10 minutes.

Second-direction SDS-PAGE electrophoresis

1. Prepare a 12% acrylamide gel.

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

3. Formulating the strip balance buffer I

4. Place a dry thick filter paper on the table, and focus the glue on the dry filter paper with the glue side facing up. Soak another thick filter paper with MilliQ water, squeeze out excess water, and then directly place on the strip, gently absorb the mineral oil and excess sample on the strip, which can reduce the vertical stripes appearing during gel dyeing. .

5. Transfer the strip to the sample hydration tray, add 6 ml (17 cm IPG) equilibration buffer I, and shake slowly for 15 minutes on a horizontal shaker.

6. Formulate Strip Balance Buffer II.

7. After the first balance is complete, remove the strip and place it on the filter paper to drain excess liquid into Balance Buffer II and continue to shake slowly on the horizontal shaker for 15 minutes.

8. Using a filter paper, remove excess liquid from the glass plate above the SDS-PAGE gel and place the dichroic gel on the table with the top of the gel facing you.

9. Heat the agarose sealant to dissolve.

10. Add TGS running buffer to the 100ml graduated cylinder.

11. After the second balance is over, remove the strip and use a filter paper to remove excess balance solution (the strip is placed on the filter paper to avoid loss of protein or damage to the gel surface).

12. Hold one end of the strip with tweezers so that the surface is completely immersed and rinsed several times in 1X running buffer.

13. Place the back of the strip toward the glass and gently place it on the long glass and add a low-melting agarose sealant.

14. Using a film of the appropriate thickness, gently push the strip down so that it is in full contact with the polyacrylamide gel. Note: Do not create bubbles under the strip. Push the support film on the back of the gel and do not touch the surface.

15. Leave for 5 minutes to allow the low melting agarose sealant to set.

16. Turn on the two-way electrophoresis condenser and adjust the temperature to 15 °C.

17. Transfer the gel to the electrophoresis tank, add the running buffer, turn on the power, and start with a low current (5mA~10mA/gel/17cm). After the sample is completely out of the IPG strip, concentrate it into a line. After that, increase the current (20-30mA/gel/17cm) and stop the electrophoresis when the bromophenol blue indicator reaches the bottom edge.

18. After the electrophoresis is finished, gently pry open the two layers of glass, remove the gel, and cut the corners for marking (wearing gloves to prevent contamination of the rubber surface).

19. Perform dyeing.

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