Hallo to everybody,
I have a question about the possibility to remove BSA from the cell medium. Has anyone tried to do it by TCA/acetone precipitation? Are there any specific protocols or may I use the 10%TCA/ice cold acetone, as used for albumin depletion in animal/human serum?
Thanks in advance!
Thanks Miguel.
Actually my aim is to identify a phospholipase in medium from hepatocytes culture which are specifically stimulated.
Because I have no clear idea whether the enzyme can be carried by albumin in cell medium as well as in serum, I was thinking to use the TCA/precipitation. It is easy and practical method, but I have not tried it before on cell medium, that is why I was asking for some suggestions.
I have also heart about the low specificity of Cibacron Blue; anyway, the big problem I should overcome is to reduce in almost "specific" way the albumin, since BSA is added to the medium (at 1%) and it invalidates protein concentration estimation.
Finally, I would like to detect the protein by WB and blindly I have tried it without purification, but even the high specificity and sensibility of the antibody, I could not see nothing.
Any advice?
You are right, Ali,
we have already tried it for mouse sera, but we have no left.....anyway, it is great to hear from others that they find the ProteoMiner good. It would be surely my next choice!
Thanks!
Indeed!
The WB has to be just a first step to assume the presence of the protein in cell medium. We have already characterized the enzyme in mouse and human sera by 2DE and MALDI MS.
Anyway, Miguel, thank you really a lot for your suggestions, they are very exhaustive and complete. They will help me a lot!
I am treasuring them, and I will do my best for it. I will keep you guys informed...
Hi Maria,
did you find a solution to get rid of BSA from the cell culture medium? I am just starting to try now. Acetone precipitation does not make you get rid of the albumin, it just concentrates and desalt the sample, (albumin included). I´m using the pierce kit for BSA depletion. So far not much success.....so, if you have any suggestion it would be great!
Hello, I just came across you r question as I also thought about a way to get rid of the BSA ......and this might be very stupid idea but I thought about filtering Serum before adding it t the Medium ... with a 5kDa cut off filter......
Or did you find by now a good and relatively easy way ?
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