Experimental reagent
Sample extract (8M urea, 2% non-ionic detergent NP-40, 2% Ampholin, pH 3.5-10, 2% DTT)
Mother liquor (30% of 29/1 Acr/Bis), ampholytes
Anode buffer 1M phosphoric acid
Cathode buffer 1M sodium hydroxide
Fixative solution (10% trichloroacetic acid, 1% sulfosalicylic acid)
Staining solution (35% ethanol, 10% glacial acetic acid, 0.15% Coomass Brilliant Blue R250)
Decolorization solution (35% ethanol, 10% glacial acetic acid)
Laboratory equipment
High-pressure electrophoresis apparatus, IEF tank, centrifuge, etc.
Experimental procedure
Sample extraction
1) 1ml prokaryotic expression cell fluid is centrifuged to take precipitation
2) The pellet was lysed with 100 ul of IEF sample buffer and centrifuged to remove the supernatant.
2. Making glue
1) Installation of ultra-thin glue device
2) Add the following reagents in sequence
Mother liquor 2ml
Urea 8M / degassing
NP-40 0.2ml
Ampholyte 2ml
10% persulfate amine 50ul
TEMED 5ul
3) pouring glue
3. Electrophoresis
1) After the pre-electrophoresis gel is solidified, the glue-making device is disassembled, and the glue is placed on the horizontal electrophoresis tank together with the support film (required, the electrophoresis tank panel is first infiltrated by the liquid paraffin, and there is no bubble between the gel support membrane and the electrophoresis tank panel) ).
2) Place the electrode strips, which are respectively infiltrated by the anode and cathode buffer solution, on the surface of the rubber surface to be in contact with the yin and yang electrodes, cover the electrophoresis tank cover, connect the power supply, and pre-electrophoresis at 200V for 10 minutes.
3) Electrophoresis, cut the tissue paper into small pieces, gently place it on the prepared rubber surface, take 10-15ul sample liquid, place on the thin cotton paper, cover the cover, connect the power supply for electrophoresis.
4) Electrophoresis parameters
200V 30min, 400V 30min, 800V4hr
4. Dyeing
1) Fix, place the electrophoresis glue together with the support film in the fixative solution for 10 min.
2) Color development, at 50 ° C, the glue and the support film are placed in the dyeing solution, and the color is developed until the band appears.
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