Hi,
So I do a lot of co-IP's and my bait protein has a molecular weight of 56kDa and its a real problem because the antibody heavy chains always seem to interfere. I have tried using clean blot to avoid this issue but it has not been a very satisfying experience so far. I always have to incubate with the regular secondary antibodies after the clean blot incubation. Does anyone have any suggestions about how to over come this problem ? My workflow with IP's is once I am done with the beads / antibody / lysate incubation - I wash the beads with LysisBuffer 3-4 times and then in the last step add Laemmli buffer. Before loading the gels, I boil the samples at 95deg C for about 5 - 7 mins.
Your suggestions will be highly appreciated ! Thank you
Hi. if you're using any reducing agents such as 2-mercaptoethanol (2-ME) in your sample buffer will results in the dissociation of heavy chains into the sample. I think Laemmli buffer itself contains 2-ME, hence use a different sample buffer for your protein sample. Hope it helps.
No need to boil the samples for more than 5 minutes in 95 C.
3-5 minutes worked well for me.
If you upload your WB picture might be helpful.
Hi,
you could try to use TreuBlot from Rockland... the rabbit and goat work quite well, the mouse is not so great and you lose some signal from your monoclonal, but it's a trade off.
Other possibility is to covalently bind your Ab to beads, if you have a purified Ab. You can choose among a range of activated beads that give rise to different types of bonds.
Good luck.
What you can also do is use an antibody conjugated to Protein A/g beads. This way when you boil the samples, there won't be any heavy chains to interfere in your western blot
All the best
thank you ! I will try out with these suggestions.. I usually use magnetic beads for IP. previously I was using sepharose beads but the results were dirty.. it was picking a lot of random stuff.. and so i was getting un-specific signals..
Hi,
Don’t use Prey and Bait antibodies raised from same species for pull down and detection. You can use antibody raised in rabbit to immunoprecipitate prey and for detection, you can go for bait antibody raised in mouse (or vice versa). Theoretically, you may find minimal cross reactivity in rabbit and mouse IgG hence less interference from heavy or light chain signal for bait detection.
Hope this may be helpful. All the best.
Hi. Sorry I should have provided this earlier.. but here is a the normal case scenario of what happens :(
In my experience, IPs using phospho-antibodies (unless they are gene specific like p-ERK1/2) give a lot of background. Can you try reversing the sequence ? Pull down Lyn and use pY to probe the blot?
Regards,
Atul
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