IgG heavy chain from IP - (May/05/2013 )

Hi, I have done a few immunoblots and i'm trying to understand why I'm getting several bands: I have done an IP of 1 specific protein, then I run this sample on a gel and transfer. Then i incubate with primary antibody, which is the same antibody as I did the IP with (this is a control lane), as I'm blotting against the same protein as the IP'd protein. it is a rabbit monoclonal antibody and i then incubate with secondary anti-rabbit IgG. I have spoken to a girl in the lab and she says it is the heavy (and a faint light) chain of the IgG. I was wondering if anyone could shed any light on why this would show up on the blot/if there is any literature on this.

Many thanks

So - a normal western detection works like this with regards to the antibodies: protein>primary antibody>secondary antibody OK?

So when you do an IP you are pulling down with an antibody and removing the protein and bound antibody from the beads - this is then denatured and run on a gel - so the antibody is still there in the form of heavy and light chains, now the secondary antibody you use during the detection can bind to those (the primary shouldn't).... If you want to get around this you need to use a primary from a different species to the one you did your IP with. You can also try using protein G-HRP conjugates in the place of a secondary, as these should bind well to the primary antibody.

Hi

so there is goat G HRPor mice or rabbit?

Also waht is the eaiest way to do a control to see where exactly is the Heavy and Light chain.

thanks

Protein G does not have species specificity other than it works better for mouse and rabbit, but it should still detect non-denatured goat IgG (i.e. intact antibodies). It shouldn't detect denatured antibodies.

see here

As bob said,

When you load the sample generated by an IP you load both your protein and the antibody you used to perform the IP. The antibody have the heavy chain and the light chain. If you used for example a rabbit antibody for the IP and the antibody you are using for the WB is an anti-rabbit you'll detect both chains of the antibody.

Take into account that the size of the heavy chain is aproximatedly 50 KDa and the size for the light chain is 20-25 KDa I think. Depending of the size of the protein you're trying to detect and the % of the gel you're using you may not see the bands corresponding to the IP antibody.

I hope this helps.

Wouldn't it be possible to use a crosslinker to crosslink the antibody to the beads? And then elute the target protein while the antibody stays rosslinked to the beads? There are commercially available kits that claim this works,and in theory it seems interesting to me. But maybe there is contamination in pratice, I have not tried it myself...

I have also seen kits utilizing beads with different functional groups that can be activated to strongly bind protein/antibodies, i.e. no protein A/G.

Possibly the yield is lower when using some elution buffers?

EDIT: E.g.  http://www.piercenet.com/media/IP-compare-all-950px.jpg   and more info http://www.piercenet.com/guide/selection-guide-immunoprecipitation-kits

claim that such kits can be used to avoid antibody elution, but then again this is commercial info... (I hope links to commercial site info is ok here?)

Yes, it works, but is typically quite expensive to do (you need quite a lot of antibody as well as the kit and beads).  It also lowers the sensitivity of the antibody as they can cross-link in any configuration.  For most IPs it isn't a problem having heavy chain present, so most people don't cross-link.

so if my ip antibody is  IgG1  it will not bind the protein A beads?

mybioforumaccount on Fri Jun 6 01:47:40 2014 said:

Wouldn't it be possible to use a crosslinker to crosslink the antibody to the beads? And then elute the target protein while the antibody stays rosslinked to the beads? There are commercially available kits that claim this works,and in theory it seems interesting to me. But maybe there is contamination in pratice, I have not tried it myself...

 

I have also seen kits utilizing beads with different functional groups that can be activated to strongly bind protein/antibodies, i.e. no protein A/G.

 

Possibly the yield is lower when using some elution buffers?

 

 

EDIT: E.g.  http://www.piercenet.com/media/IP-compare-all-950px.jpg   and more info http://www.piercenet.com/guide/selection-guide-immunoprecipitation-kits

claim that such kits can be used to avoid antibody elution, but then again this is commercial info... (I hope links to commercial site info is ok here?)

i've used the cross-linking kits. the ones i've used worked very well, and the antibody bound at the fc region so they were fully functional (or, at least, appeared to be, i didn't really check).

the one i used was developed by s&s but was eventually sold to pierce. you can look at it on this webpage. look at this page for an ip kit with antibody crosslinking.