I am optimising ChIP for my protein which is approx. 45kDa in size. Upon doing Western Blot to check my pulldown I see a big IgG band that covers up my protein. Can anyone suggest me how to resolve this? qPCR is another option but I want to first see the pulldown by Western blotting.
Also, can some one suggest me secondary antibodies that do not recognise the IgG have chain that I can use to avoid this situation?
You could perhaps try the IgG purification kit. I have used this before, but the overall yield is reduced drastically. But you could give this a try.
http://www.piercenet.com/product/melon-gel-igg-purification-kits
Good luck,
Chandana
You can try the suggestion from Katrin, even so in my experience, light-chain specific secondary NEVER worked well :) Whatever salesmen tells you, in reality such AB always gave strong cross-reactivity with heavy chains. However, another thing you can do is to simply remove DTT or beta-mercapto (whatever you are using) from the sample buffer and boil your samples for a shorter period of time. In this case, most of your AB do not brake into light and heavy chains (partially they still do, just due to the heating, but to much lesser extent) and you have much higher chances to resolve your protein of interest from IgGs.
One more option is to use protein A conjugated with HRP, because protein A does not bind to denatured IgGs, so, in theory, you should not see any traces of the AB you used to IP your protein of interest, but only secondary AB.
You can try "Native page" by using native page sample buffer without mercaptoethanol or DTT for electrophoresis. IgG will show an approx 160 kDa band at the top of the gel.
native page sample buffer
4 ml dH2O,
1 ml 0.5 M tris-HCl (pH 6.8)
0.8 ml glycerol,
1.6 ml % 10 SDS,
0.2 ml % 0.05 bromophenolblue
- Badges
- Science method