3 replies to this topic

[PhD]

Hello!

I was just wondering whether its going to be a problem to send away my induced fragments after theyve been denatured with urea since only then do I remove them from the pellet. Are there any tricks I can use to increase the fragments antigenic properties. Someone suggested its better to send away a gel cut out?

Thank u i advance for any replies!!

[K.B.]

Yes, it can make a difference - you can produce antibodies against epitopes that are not "visible" in native protein.

In my opinion using protein band from SDS-PAGE instead of urea denatured protein makes no sense, since SDS-PAGE is even more denaturing because it's using strong ionic detergent (SDS) and disulfide-breaking reagent(s) (eg. DTT, bME).

You may try to re-nature your protein eg. by rapid dilution. (Three-phase partitioning (TPP) has also some re-naturing properties - contact me for details if you want to try this method).

[little mouse]

It depends what you want to do with your antibody. If you want to do western-blot on denatured proteins, it's fine. If you want to do ELISA or Flow cytometry, it won't be fine, for the reasons K.B. told you.

[PhD]

Uhm I see, I do need specific ones. well I have some fragments that are somewhat soluble under native conditions but most Im going to have to denature with Urea. I guess I will try refolding.

Thank u for input