Immunoprecipitation - light chain and heavy chain - (Jun/16/2006 )

I have done one immunoprecipitation using Ezyview red protein G Affinity Gel (Sigma), targeting on protein with size 34kDa. I can see the heavy chain at around ~55kDa.
Now I'm worrying if the band at ~34KDa is actually my target protein or is it an IgG light chain, because the bands are quite fat. Does IgG light chain appear on that size? Because from all the literature they said it is around 20kDa.

Also how to block for the IgG binding, so you won't mistakenly taking the IgG band for the real band.

My antibody itself is raised in rabbit.

Hope anyone can help me with this.

I would scan the film first of all.
I would then strip and probe with another Ab: if you still have the sme band it's the IgG, otherwise it was your protein. The light chain is anyway around 20-25. The alterative is that next time you do a no Ab IP and that you also load 1ug Ab in another lane.

If you prepare crosslinked bead sets, you won´t see any IgG chain in your Western Blott. It permits you also to reuse them after proper regeneration. The only problem is that they don´t have the same capacity, you´re gonna allways loose some activity.

Which kind of antibody is yours? Is it affinity purified?

It is not an affinity purified, just a normal rabbit polyclonal IgG antibody.

So how do you prepare a crosslinked bead sets?

Let me know wether you received the protocol, I´m not still very familiar with the personal account of the forum

Miguelon

there are different thinks you can do to avoid the detection of denatured Ig :
-use crosslink bead sets
-detect with protein A or G coupled to HRP
-use biotinylated primary antibody, and streptavidin HRP for detection

Hi Guys,

Miguelon, yes i've received your protocol, thanks! I'll look into it, and if there's more problem i'll contact you.
Missele, thanks for the suggestions too....
Will contact you guys when i have more problems...