Two-dimensional electrophoresis complete operation steps
2021-05-06 08:02:30
(1) First-direction isoelectric focusing
1. Take a small tube (1 ml/tube) of hydrated loading buffer (I) (without DTT, without Bio-Lyte) frozen at -20 °C from the refrigerator and dissolve at room temperature.
2. Add 0.01 g DTT, Bio-Lyte 4-6, 5-7 each 2.5 ml to the small tube and mix well.
3. Remove 400 ml of hydration loading buffer from the vial, add 100 ml of sample, and mix well.
4. Take a -20 °C cryopreserved IPG pre-formed strip (17 cm pH 4-7) from the freezer and leave it at room temperature for 10 minutes.
5. Add the sample linearly to the left and right along the edge of the groove in the focus or hydration disk. Do not add about 1cm at each end of the groove, and the sample liquid in the middle must be continuous. Note: Do not create bubbles. Otherwise it affects the distribution of protein in the strip.
6. When all protein samples have been added to the focusing tray or hydration tray, gently remove the protective layer from the prefabricated IPG strip with tweezers.
7. Divide the positive and negative electrodes of the strip, and gently place the IPG strip face down on the sample solution in the focusing tray or hydration tray, so that the positive electrode of the strip (marked with +) corresponds to the positive electrode of the focusing disc. . Make sure the strip is in close contact with the electrode. Do not get the sample solution onto the plastic support film on the back of the strip because these solutions are not absorbed by the strip. Also be careful not to create bubbles in the solution below the strip. If air bubbles have formed, gently lift one end of the strip with tweezers and move the strip up and down until the bubbles are caught outside the strip.
8. Cover each strip with 2-3ml of mineral oil to prevent evaporation of the liquid during the hydration of the strip. Slowly add mineral oil and slowly add mineral oil to the plastic support film along the strip.
9. For positive and negative, cover. Set the isoelectric focusing program.
10. Focus on the finished strip. Immediately equilibrate, second-direction SDS-PAGE electrophoresis, otherwise the strip is placed in the sample hydration tray and stored in a -20 °C refrigerator.
(2) Second-direction SDS-PAGE electrophoresis
1. Prepare two pieces of 10% acrylamide gel. Equipped with 80ml gel solution, 40ml per gel, the solution was injected into the glass plate sandwich, leaving 1cm space in the upper part, and the n-butanol cover was saturated with MilliQ water, ethanol or water to keep the rubber surface flat. Aggregate for 30 minutes. After the general gel is layered with the upper liquid, it indicates that the gel has substantially polymerized.
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. Remove the strip from the -20 °C freezer and let it dissolve for 10 minutes at room temperature.
4. Prepare the strip balance buffer I.
5. Place a dry thick filter paper on the table, and focus the glue on the dry filter paper. Dip another thick filter paper with MilliQ water, squeeze excess water, and place directly on the strip to gently blot the mineral oil and excess sample on the strip. This can reduce the vertical streaks that occur when the gel is dyed.
6. Transfer the strips to the swell tray, one strip per tank, and add 5 ml strip balance buffer I to the tank with the strip. Place the sample hydration tray on a horizontal shaker and shake slowly for 15 minutes.
7. Prepare Strip Balance Buffer II.
8. After the first equilibration, completely pour or aspirate the strip equilibration buffer I in the sample hydration tray. Use a filter paper to absorb excess balance solution (pose the strip on the filter paper to avoid loss of protein or damage to the gel surface). Add Strip Balance Buffer II and continue to shake slowly on a horizontal shaker for 15 minutes.
9. Use a filter paper to remove excess liquid from the glass plate above the SDS-PAGE polyacrylamide gel. Place the treated second-direction gel on the table with the long glass plate underneath, the short glass plate facing up, and the top of the gel facing you.
10. Heat the agarose sealant to dissolve.
11. Dilute 10× electrophoresis buffer and dilute the cartridge by 10 times into 1× running buffer. Catch the bubbles on the surface of the buffer.
12. After the second equilibration, completely pour or aspirate the strip equilibration buffer II in the sample hydration tray. Use a filter paper to absorb excess balance solution (pose the strip 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 and clamp the end of the strip with tweezers to completely immerse the gel in 1X running buffer. The glue is then placed face up on the long glass plate of the gel. The remaining strips operate in the same way.
14. Transfer the SDS-PAGE gel with the strip to the potting rack with the short glass facing you. A low melting agarose sealant was added over the gel.
15. Using a pair of tweezers, tongue depressors, or flat-headed needles, gently push the strip down so that it is in full contact with the polyacrylamide gel. Be careful not to create any air bubbles under the strip. When pushing the strip with tweezers, tongue depressors or flat needles, be careful to push the support film on the back of the gel, not touching the rubber surface.
16. Leave for 5 minutes to completely solidify the low melting point agarose sealant.
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, use low current (5mA/gel/17cm) or low voltage at the beginning, wait until the sample is completely out of the IPG strip, concentrate it into a line, and then add High current (or voltage) (20-30mA/gel/17cm), the electrophoresis can be stopped when the bromophenol blue indicator reaches the bottom edge.
19. 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).
20. Perform staining.
1. Take a small tube (1 ml/tube) of hydrated loading buffer (I) (without DTT, without Bio-Lyte) frozen at -20 °C from the refrigerator and dissolve at room temperature.
2. Add 0.01 g DTT, Bio-Lyte 4-6, 5-7 each 2.5 ml to the small tube and mix well.
3. Remove 400 ml of hydration loading buffer from the vial, add 100 ml of sample, and mix well.
4. Take a -20 °C cryopreserved IPG pre-formed strip (17 cm pH 4-7) from the freezer and leave it at room temperature for 10 minutes.
5. Add the sample linearly to the left and right along the edge of the groove in the focus or hydration disk. Do not add about 1cm at each end of the groove, and the sample liquid in the middle must be continuous. Note: Do not create bubbles. Otherwise it affects the distribution of protein in the strip.
6. When all protein samples have been added to the focusing tray or hydration tray, gently remove the protective layer from the prefabricated IPG strip with tweezers.
7. Divide the positive and negative electrodes of the strip, and gently place the IPG strip face down on the sample solution in the focusing tray or hydration tray, so that the positive electrode of the strip (marked with +) corresponds to the positive electrode of the focusing disc. . Make sure the strip is in close contact with the electrode. Do not get the sample solution onto the plastic support film on the back of the strip because these solutions are not absorbed by the strip. Also be careful not to create bubbles in the solution below the strip. If air bubbles have formed, gently lift one end of the strip with tweezers and move the strip up and down until the bubbles are caught outside the strip.
8. Cover each strip with 2-3ml of mineral oil to prevent evaporation of the liquid during the hydration of the strip. Slowly add mineral oil and slowly add mineral oil to the plastic support film along the strip.
9. For positive and negative, cover. Set the isoelectric focusing program.
10. Focus on the finished strip. Immediately equilibrate, second-direction SDS-PAGE electrophoresis, otherwise the strip is placed in the sample hydration tray and stored in a -20 °C refrigerator.
(2) Second-direction SDS-PAGE electrophoresis
1. Prepare two pieces of 10% acrylamide gel. Equipped with 80ml gel solution, 40ml per gel, the solution was injected into the glass plate sandwich, leaving 1cm space in the upper part, and the n-butanol cover was saturated with MilliQ water, ethanol or water to keep the rubber surface flat. Aggregate for 30 minutes. After the general gel is layered with the upper liquid, it indicates that the gel has substantially polymerized.
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. Remove the strip from the -20 °C freezer and let it dissolve for 10 minutes at room temperature.
4. Prepare the strip balance buffer I.
5. Place a dry thick filter paper on the table, and focus the glue on the dry filter paper. Dip another thick filter paper with MilliQ water, squeeze excess water, and place directly on the strip to gently blot the mineral oil and excess sample on the strip. This can reduce the vertical streaks that occur when the gel is dyed.
6. Transfer the strips to the swell tray, one strip per tank, and add 5 ml strip balance buffer I to the tank with the strip. Place the sample hydration tray on a horizontal shaker and shake slowly for 15 minutes.
7. Prepare Strip Balance Buffer II.
8. After the first equilibration, completely pour or aspirate the strip equilibration buffer I in the sample hydration tray. Use a filter paper to absorb excess balance solution (pose the strip on the filter paper to avoid loss of protein or damage to the gel surface). Add Strip Balance Buffer II and continue to shake slowly on a horizontal shaker for 15 minutes.
9. Use a filter paper to remove excess liquid from the glass plate above the SDS-PAGE polyacrylamide gel. Place the treated second-direction gel on the table with the long glass plate underneath, the short glass plate facing up, and the top of the gel facing you.
10. Heat the agarose sealant to dissolve.
11. Dilute 10× electrophoresis buffer and dilute the cartridge by 10 times into 1× running buffer. Catch the bubbles on the surface of the buffer.
12. After the second equilibration, completely pour or aspirate the strip equilibration buffer II in the sample hydration tray. Use a filter paper to absorb excess balance solution (pose the strip 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 and clamp the end of the strip with tweezers to completely immerse the gel in 1X running buffer. The glue is then placed face up on the long glass plate of the gel. The remaining strips operate in the same way.
14. Transfer the SDS-PAGE gel with the strip to the potting rack with the short glass facing you. A low melting agarose sealant was added over the gel.
15. Using a pair of tweezers, tongue depressors, or flat-headed needles, gently push the strip down so that it is in full contact with the polyacrylamide gel. Be careful not to create any air bubbles under the strip. When pushing the strip with tweezers, tongue depressors or flat needles, be careful to push the support film on the back of the gel, not touching the rubber surface.
16. Leave for 5 minutes to completely solidify the low melting point agarose sealant.
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, use low current (5mA/gel/17cm) or low voltage at the beginning, wait until the sample is completely out of the IPG strip, concentrate it into a line, and then add High current (or voltage) (20-30mA/gel/17cm), the electrophoresis can be stopped when the bromophenol blue indicator reaches the bottom edge.
19. 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).
20. Perform staining.
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