Once you have done the pull down followed by western blotting. You need to develope the blot with primary and secondary antibody. Follow general WB protocol. You will see the bands in Input as well as in Immune or Ab pull down fraction. IgG will work as negative control. It does not matter which beads you had used. Both magnetic bead and protein G sepharose bead works.

Destain your ponceau stained nitrocellulose membrane and probe with primary antibody followed by secondary antibody and develop the blot similar to a routine WB.

What concerns me in your case as assessed by your ponceau stained membrane is that your protein of interest seems to be close in molecular weight to the IgG heavy chain. If that is true then you will have to change your IP protocol to include a step on crosslinking your antibody to the beads. This will get rid of the IgG heavy chain band and you will only be left with your protein of interest.

Which crosslinker you suggest Avinash? Does it crosslinks the antibody with Protein-A/G or with sepharose beads?

Hi, 

please be aware that cross-linking the prim Abs to the bead surface might have an impact on the functionality of the capture antibody.

there are 2 ways to by-pass the problem of heavy and light chain leaking off the solid surface

- covalent coupling of prim Ab to e.g. Dynabeads M-270 epoxy (e.g. co-IP kit or antibody coupling kit

and/or in combination with

- TrueBlot secondary HRP conjugated Ab which will not recognize heavy or light chain on the membrane.

kind regards

ketil

Hello Niharika,

If I understand your comment, it seems that you're using the same antibody for both the IP and primary steps in your procedure.  A common problem that individuals encounter upon using a capture IP antibody of the same species as the primary WB antibody, let alone, the same antibody, is the appearance of non-specific bands due to H + L chains of your IP antibody.

To avoid this, I've used IP and detection antibodies of different species.  See if you can obtain another IP or detection antibody that recognizes your target but is of a different species, and retry your protocol.  

Best of luck!

Shannon

Dear Avinash,

Thank you so much for your reply.

I am detecting Drosha. which is 159 Kda. So there is no problem with molecular weight.

Best Niharika

Dear Shannon,

Thank you so much for your reply.

Indeed i used the same primary Ab for both coating the magnetic beads and detection.

I guess it could be one of the reason for this problem.

I tried again with stripping off and using HRP conjugated Ab (previously i used Odysee )

on exposure it is showing all the bands.

i will try your suggestion. thanks a lot