Hi! I have a technical question concerning protein extraction from neuronal primary cultures. When I grow glial cells in the wells (coated with PLL), after I aspirate the supernatant I usually wash them 3 times with clean PBS. It allows me to get rid of all the residual debris and media. After I add RIPA buffer and scrape the cells. With neurons however, I cannot use PBS as a washing solution because neurons don't like salts and when I used it once at the begging, after measuring the protein concentration, it turned out that I barely have any. So right now I am not washing the wells and add RIPA straight away, however I think that the sample/western blot quality is compromised. Do you have experience with that kind of issue?
Thanks
Hello, I suggest you to lower your PBS concentration because I do not think that salts are the main reason to not get any protein concentration (even though you mentioned that neurons do not like salts). When you added RIPA straight away, how could you tell that it is the real reason why you have a compromised quality of your western blot detection? I am asking this because the washing step before or after cells harvest can differ a lot, even the number of washings to do is not fixed, so finally I think that maybe another or other reasons can be invloved in the quality of your detection or result.
Good Luck.
Washing with sterile 1 X PBS should not affect the protein of neurons or any other cells unless the PBS is not 1X or if it’s contaminated. You do not need to wash 3 times in this step, this might have lifted & washed off your cells with PBS. Did you check whether you had enough cells in the well/dish before and after rinse?
3 X washing is mainly during antibody treatments.
If you do not wash the cells and add RIPA buffer after just completely removed the culture media, still the protein will be okay. Hope you purchased the RIPA. In case if you make it, check it carefully for the composition, pH, etc.
If all you did was ok if you feel like not using 1 X PBS, do not want to use the cells without rinsing, you may add a few drops of RIPA itself & rotate the dish & remove RIPA carefully/gently & quickly from the cells. Then add RIPA, scrape the cells, and collect cell extract.
There may be so many reasons that your Western blot did not come out well. You stated you did not have enough protein, how much protein you have loaded? did you check your SDS-PAGE while running, before and after transfer? was the transfer complete? did you use a protein ladder? you can upload here the pictures of cells before harvesting, SDS-PAGE & WB.
Thank you for the answers!
As the follow up, to fully understand the situation, the western blots I am running are "fine" but if I want to compare the quality of samples, that I see on WB, in which the cells were washed with PBS before RIPA (as I do with glial cells) I can see that washed cells are more "neat". But I still see the signal in both gels, so it's not the WB problem. I was only wondering if someone is washing the neurons with something different than PBS that would not compromise the cells/protein concentration.
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