Good morning, I am analyzing a protein after a treatment in hypocotyls by WB. After 3 days postagroinfiltration, I take my seeds and extract protein with the Pierce plant total protein extraction kit following the manufacturers protocol. However, when I load 30 ug of the sample into a gel and then Coomassie staining, I see a smear that I think is the result of protein degradation. I attach a photo of the Coomassie gel that I see, do you usually have similar results? How can I confirm that I have protein degradation? Also, after incubating with my antibody, and revealed by ECL, the signal that I obtain has too much noise and with no evident bands in the weight that I expect (Also attached). Can anyone give me a pice of advice with my problem? Thanks in advance! Best,
It's been a while since I did any SDS-PAGE and Western blot, but
1) the gel seems OK to me. Coomassie staining is not specific, so you should expect many bands, which may fuse together
2) with a little bit of fantasy, the Western blot show some weak bands. Maybe just your protein is present in very low amounts? Can you try to tweak the conditions of your blotting?
1. On your SDS-Page, the last band of protein marker just reaches the middle of the page, which may not separate your target protein effectively. It would help if you prolonged the time of SDS- Page electrophoresis.
2. You can attempt to purify your target protein from total protein to increase its proportion， such as affinity chromatography, molecular sieve chromatography, and immunoprecipitation.
3. if your western blot system is ok, you can fully separate the total protein by two—dimensional gel electrophoresis， and then detect your target protein by western blot.
https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0018670_PiercePlantTotalProteinExtractionKit_UG.pdf
Low protein activity Extract is degraded. Keep sample cold and use protease and/or phosphatase inhibitors.
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