Hi!
I have purchased a CD34-FITC (IgG) antibody from a company and in order to conjugate it with a gold nanoparticle, I had to purify it and remove all the stabilizing proteins. I used a BSA removal Kit from abcam for this. After purification I ran a non-reducing SDS PAGE and I could still see a band at about 55 kDa. I am not sure if this is still leftovers impurities and I need to improve the purification protocol or maybe it is the heavy chain of my Ab and the band shows some kind of dragradation. Does anyone have experience with this and could help?
Thank you in advance!
Dear Daniela
Everything is possible but considering the high amount of BSA that was present before purification, i suspect that the band is just an excess of BSA that the kit was not able to remove and that you will remove it by retreating the sample with BSA removal kit.
ciao
Manuele
Dear Daniela Lascu
All your doubts are possible.
Why can't you try western blotting which giver you little clarity about few of your doubts. As it gives specificity as well as purity. If you get multiple band in western blot then its degradation if you get single band then its impurities
I think BSA is not removed completely by the removal kit. BSA is around 66 kDa. A difference of approx. 10 kDa. might be due to fact that you are using non-reducing conditions. You can run reducing PAGE to know the molecular weights of antibody fragments.
This article link may be helpful.
Article Unfolding of IgG domains detected by non-reducing SDS-PAGE
Could be BSA carry over. If it is Ab then you could WB with antibody against CD33 species? Could be heavy chain.
Is your Ab visible at ~160 kDa? There should be proportionally way more of your un-reduced Ab than BSA. If the only band you see is @ ~55 kDa, most likely BSA.
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