My protein of interest is a polymeric antibody (>300 kD). I would like to probe the polymeric antibody with a reagent (a recombinant protein in a supernatant) that can only bind to the J-chain (with the antibody quaternary structure intact). My first try running purified antibodies from a commercial source on a Biorad Mini Protean TGX 4-20% gel (without adding 2-mercaptoethanol or heating in sample prep) didn't work (got splotchy blobs and bands that don't make sense). I have used the same antibodies in ELISA before and they worked. Since the Bio Rad website says the gel I used can also be used for a native PAGE, I am not sure what else to do differently. Any suggestions?
use a lower percentage stacking gel
4% stacking and 7 % resolving......
I reckon 4% Tris-Glycine from lifetechnologies
https://www.lifetechnologies.com/order/catalog/product/EC60585BOX?ICID=search-product
For native proteins please have look at the below image.
https://www.lifetechnologies.com/order/catalog/product/BN1003BOX
I prefer LifeTechnologies gels over BioRad gels for better resolution and tight bands.
Thanks, so just a lower percentage gel and run the gel similar to an SDS PAGE (without the heating and 2-mercaptoethanol)? We have a Biorad apparatus, not sure if the lifetechnologies would be compatible... or is it?
You would have to run a native gel. There are special sample buffers (without SDS) that have to be used to run samples in these gels. Life technologies has one such sample buffer.
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