Hi,
Yesterday i was purifying biotinylated molecules with a neutravidin polyacrylamide resin and after incubation with cell lysate, the resin looked "aggregated" instead of a conventional resin suspension.. After extensive PBS 0,5M and PBS washes, it keeped in the same way.
Do you know why this happens?
Thanks for reading
Martin
Do the biotinylated molecules have several biotin residues? How many binding sites does neutravidin have?
Hi there,
It sounds like protein precipitation occuring when applying lysate onto the resin. Do you equilibrate the resin with the lysis buffer? How concentrated is your cell lysate? Is your cell lysate properly cleared from unsoluble material? I would suggest to dilute the cell lysate prior to application to the resin.
Hi Martin, neutravidin can bind 4 biotins and if your protein has more than 1 biotin you are seeing crosslinking between the biotinylated protein and the neutravidin resin.
Thank you for the answers,
Omar, NeutrAvidin (like Avidin also) resin has four binding sites and molecules have probably several biotin residues
Dominique, the resin was equilibrated with lysis buffer and the lysate was cleared form unsoluble material. I did not know the protein concentration. The lysate came from 5 million mouse fresh splenocytes.
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