Hello everybody, I did already 3 times the protein extraction through RIPA lysis buffer, and when I load my samples I continue to obtain this result. I am sure it's a problem during protein extraction because I tried with another sample from my colleague and it went fine. What can I do wrong? I scrape cells, add PBS, centrifuge, discard the supernatant, resuspend the pellet in cold PBS. I do this twice and then I resuspend my pellet in RIPA buffer + protease inhibitor, vortex time to time and leave on ice for 30 minutes, then spin max speed for 30 second, transfer the supernatant in a new Eppendorf, quantify with BCA protocol, mix the right amount of proteins and Laemmi Buffer, heat at 95° for 5 minutes, put in ice, spin and load.
Any suggestion?
I attach the picture.
Possibly the composition of your RIPA buffer is incorrect, with too much detergent.
Dear Alessandra Macaluso
I would suggest that the SDS-PAGE gel you shared with us could be explained by either a pH problem or a lysis problem (more likely)
First: you need to check the pH of your RIPA buffer (it should be 7.4 or 8), also the pH of the running buffer. if both as you are suppose to be, then you can eliminate the pH.
Second: you mentioned that you only vortexing your samples after adding RIPA (vortex time to time and leave on ice for 30 minutes, then spin max speed for 30 second) how was the viscosity of your samples after this quick spin? As you know, if the lysing was not done correctly, samples will have nucleic acids (DNA/RNA) and other cell debris along with the solubilized proteins. Of course having these contaminates will affect the electrophoresis.
I suggest you to choose one of these methods to lyse your samples and shear the nucleic acids: 1. sonication, 2. incubate samples in RIPA for 30 min with vigorous shaking at 4 C, 3. homogenization using homogenizer or needle and syringe. After lysing the samples you will need to clear the supernatant with centrifugation for 10-20 min at >10,000 rpm at 4C.
Best of luck
Rabab
It could be viscosity problem or overload of protein. How was your collegue sample the same or more viscuous as yours? ANd what abour the amount of protein you are loading?
Then II have a question : why do you place your samples on ice after adding laemli and heating? TIn my opinion, the SDS contained in you laemli buffer will then precipitate ans can also induce migration troubles.
I know all this does not help you much but if it can help finding a reason for this...
Good luck
Anne-E
- Badges
- Science topic