I need to collect proteins after SDS-PAGE electrophoresis to quantify the proteins present in the target bright line using the bradford method. So how can I separate the protein and remove the polyacrylamide gel and SDS from the protein? Can you recommend some methods and ways to do that? Thanks for everything.
the old fashioned method was to cut the gel slice and tie it into a short length of dialysis tubing with some electrophoresis buffe. Drop the tubing into the e/f tank and turn on the current so the protein leaves the gel and goes into the buffer, The dialysis tubing must be of a grade that keeps the protein inside it. Then just pipette the buffer and protein from the tubing leaving the PAG behind
Hello Hoàng Trần
You may use the method of passive elution of protein from polyacrylamide gel pieces. The protocol is as follows.
1. You may place the cut gel pieces containing the protein of interest in a clean microcentrifuge tube.
2. Add 0.5−1 ml of elution buffer (containing 50 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, pH 7.5) in such a way that the gel pieces are completely immersed.
3. You may crush the gel pieces using a clean pestle, and incubate overnight on the shaker at 30°C.
4. Centrifuge at 5,000−10,000 x g for 10 min, and carefully pipet the supernatant into a new microcentrifuge tube.
5. You may confirm the presence of protein by subjecting a portion of the supernatant to SDS-PAGE.
6. If you wish to concentrate your protein of interest, you could use the acetone precipitation method to concentrate the protein.
Best.
https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/extract-proteins-polyacrylamide-gels-tech-tip.pdf
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