Surface proteins isolated by biotinylation are contaminated.. - (May/31/2007 )
I had isolated transfected surface proteins from HEK293 cells by biotinylation using Pierce's sulfo-NHS-SS-biotin with good success. No internal protein (tubulin) contamination at all. However, my recent attempts of repeating the experiments using the same system before ended up with surface proteins mixed with internal proteins. I changed all the buffers and used the freshed stock of SS-biotin or LC-biotin. Still no luck. It's getting very frustrating since this experiment becomes main tool of my project. I tried Biotinylation kit from Pierce and GE, and was not happy with the results, always contaminated with tubulins. I use NaKATPase as marker of plasma membrane proteins and tubulin as internal proteins.
Is anyone who can help me out, please ?? Thanks a lot !!
Is anyone who can help me out, please ?? Thanks a lot !!
May have problem at the washing or incubation steps. Biotinylation and washing all have to be performed either on ice (incubation) or at 4oC (wash). The washing needs to be done thoroughly, make sure that all free biotin is sequestered away before lysing cells. If you are using the biotin that is supposed to bind to NH2 group, wash 4x10min with 0.05M NH4Cl, followed by 2x10min rinse with PBSCM (all cold).
Is anyone who can help me out, please ?? Thanks a lot !!
May have problem at the washing or incubation steps. Biotinylation and washing all have to be performed either on ice (incubation) or at 4oC (wash). The washing needs to be done thoroughly, make sure that all free biotin is sequestered away before lysing cells. If you are using the biotin that is supposed to bind to NH2 group, wash 4x10min with 0.05M NH4Cl, followed by 2x10min rinse with PBSCM (all cold).
Thank you for your suggestion. Indeed, I stopped biotinylation by washing once and incubating the cells with Gly-PBS buffer for 15 min in ice before lysis. Still, it seems to be washed more thoroughly. Another thing I'm wondering is sulfo-NHS-SS-biotin solution I've prepared may have been prepared too early before adding to cells. This Wednesday I'll use the modified methods and post new results here. Thanks again !!
The final solution of biotin (to add directly to cells) should only be prepared just before use (dilute the stock, mix and add immediately to cells) since it is easily hydrolyzed. It would be best if you do biotinylation twice (15-20min each) instead on once for 30min. Each time have to be done with freshly prepared biotin solution. The washing steps are critical as it will prevent the biotinylation of intracellular proteins. Good luck.