I want to quantify proteins in serum free conditioned media. My only issue is that my media has phenol red which reacts weirdly with BCA's working reagent. I suspended my standards (BSA) in serum-free unconditioned media and used serum-free unconditioned media as my blank, but I got negative values. Is there another/better way to quantify proteins in my media?
Thanks
Why do not you use a UV spectrophotometer if you know your protein sequence/ absorption coefficient? Using a Nanodrop will be an other alternative option if you do not have access to UV spectrophotometer.
I'm not sure whether this will help with the phenol red problem, but you can try it. Precipitate the proteins in microfuge tubes using 1/10 volume of 72% trichloroacetic acid and 1/10 volume of 0.15% sodium deoxycholate (2 separate solutions). After 10 minutes, centrifuge the samples at top speed of a microcentrifuge. Carefully remove all of the supernatant. Perform the assay on the pellets. As long as there is not too much protein, the pellets should dissolve in the BCA reagent.
You might also try the Bradford assay.
Thank you for your helpful suggestions. Dr. Shapiro, I already did protein precipitation (TCA/acetone) and it works, but I am sure the process involves loosing some proteins, which I can't afford, given that my concentration is already low. So I prefer checking and comparing the concentration in the actual media.
Dr. Tekewe, I tried the nanopdrop and my values were really off. My 260/280 was 6.92 and -0.012 for my other samples and my concentrations were really low. So I tried concentrating my media, which again, concentrated the phenol red, in which case the nanodrop refused to read anything.
I ordered media with no phenol red and will let you know if it works.
Thank you for the help!
Hello Mayassa! How did u fix your problem? would u plz let me know? i have the same problem.
Hello Aysha, I ended up buying a concentrator (Amicon) and using phenol red free DMEM
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