Heavy Light Chain Immunoprecipitation Problems
 

Hello,
i have a question for IP and antibody heavy and light chain detection.


I performed an Immunoprecipitation experiment but when i ran the SDS-PAGE gel, i loaded the rabbit antibody used for ip in a control lane however
doing the western blot with Goat anti-rabbit secondary antibody only detected a very strong 50kDa band in the lane!


Did it mean that the antibody is not completely denatured? :spcartmanmad:

Should i expect another 20-30 kDa band on the gel corresponding to the antibody light chain?

I also boiled the antibody in loading buffer for 5 min so shouldnt this be ok?

Thanks again in advance

Re: Heavy Light Chain Immunoprecipitation Problems
 

Have you tried running a gel without DTT, BME, but with SDS that will not separate the heavy and light Ab chains?

Re: Heavy Light Chain Immunoprecipitation Problems
 

I'm trying to rid heavy chains from my IP' How do you think is the suitable SDS concentration to do this? Withouth DTT and BME?

Re: Heavy Light Chain Immunoprecipitation Problems
 

whole IgG=150 KD
!/2 IgG =75 KD
Heavy chain only=50 KD
Light chain=25 kD
If you only see 50 KD band. That indicates your ab is specific for heavy chain not light chain the light chain is there but you can't detecte it. Does this makes sense?