I am using a mammalian cell line (Vero) and need to infect the cells with a virus resulting in reduced cell number at the end of the experiment. I plan to standardise either the number of cells (by counting) or by performing a BCA protein assay to normalize protein concentration before I proceed with ELISA.
My question is after I homogenise the protein in PBS (the protocol use PBS as the homogenising medium) to carry out the BCA assay, can I store the remaining protein supernatant directly at -80C? Do I have to add a PMSF inhibitor? I cannot run the BCA assay and ELISA on the same day because of technical issues with the machine only available during office hours. Any ideas or technical tips are very much helpful.
Hello Aisyah Jaafar
I would recommend that you aliquot the larger lysate volume into many smaller lysate volumes. Then a few tubes you may store at -20 degree C for immediate use, while the rest you can store at -80 degree C until you need them sometime later. This will ensure that the tubes only go through a few freeze/thaw cycles before they are used so as to limit the degradation of protein. Centrifuge the lysate again before you use it for immunoassay.
Best.
I would normally add the recommended concentration of protease inhibitor cocktail in the lysis buffer to avoid degradation. I would then store the samples at -80 C until use. PMSF has solubility issues and is not very stable in the aqueous environment. The effectiveness of PMSF significantly reduces after 30 - 40 minutes.
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