I'm trying to concentrate cell culture supernatant to evaluate levels of IL-1B trough ELISA. I would like to do with TCA-Acetone method. Do you have any good protocol? Also, I've read that TCA-Acetone method could damage small proteins, so IL-1B could be damaged. Do you have any other possible methods? Many thanks for any suggestion.
Fulvio,
Ultrafiltration is a non-chemical method for protein concentration, column filtration units that work by centrifugation are available with different molecular weight cutoffs. Other alternatives might include immunoprecipitation.
Hopefully, someone has some advise for your specific application.
Good luck
The use of commercial columns is the best solution. Usually TCA-Acetone method generate a pellet difficult to resuspend and you may loose a lot of proteins.
Ultrafiltration, as suggested by Steven Larsen is a good method if the medium does not contain serum, because the serum proteins will also be concentrated. There are columns for removing serum albumin, which could alleviate this problem. TCA precipitation is also problematic if the medium has serum because you get a huge pellet that is hard to dissolve.
The ultrafiltration membrane chosen should have a molecular weight cutoff well below the molecular weight of the protein you wish to concentrate so that the protein will be retained.
Thanks for all the suggestions! unfortunately I cannot access right now to column or ultrafiltration method..this is why I'm looking for a nice TCA protocol...expecially thanks to Saleh e Nicola, suggesting nice methods...I'll try and see what happens...
Hi Fulvio, Wich method for precipitation you use finally?, I had the same problem, need concentrate proteins from supernatant and detects growth factors trowgh ELISA. Please can you help me.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA