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problems about western blot after Co-IP

problems about western blot after Co-IP - Immunoprecipitation Forum

problems about western blot after Co-IP - IP or Immunoprecipitation Forum.


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  #1  
Old 11-19-2008, 12:45 PM
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Default problems about western blot after Co-IP



Hi,ereryone

I have done my co-ip experiments without cross-link my antibody to protein A beads. So when I performed western blot to check those interacting proteins, the antibody contamination was serious. I have seen the introduction of Thermo Scientific Clean Blot Reagent on their website. They said they can solve this problem.I just feel a little bit worry about whether it is so good as the website showed. I can not find any reference which cited this product. So has anyone used this reagent before? Can you give me some information about it?
Thank you very much!

Jenny
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Old 11-20-2008, 06:42 AM
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Default Re: problems about western blot after Co-IP

Hello
welcome to the forums
do you have any pictures to show us?
there are more people to help if they can see whats wrong.
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Old 11-21-2008, 05:06 PM
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Default Re: problems about western blot after Co-IP

They list this as a new producton their website, so it won't have any published references for awhile. Also from the manufacturer's website, "Clean-Blot IP Detection Reagent detects only the native antibodies, providing accurate and specific detection of the target antigen."

So, it seems like it's based on a sound principle...heavy and light chain on the blot are denatured and won't be recognized, but the primary antibody is in its native form and will be recognized by the secondary.
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Old 11-22-2008, 09:13 AM
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Default Re: problems about western blot after Co-IP

Thank you for help. I just don't know it is a new product. If it really works so well, it will help me a lot. I don't know how to insert the figure here. I can describe my problem first.
The secondary antibody of the antibody I used to do my co-ip is anti-rabbit. I want to
check some interaction proteins by WB after co-ip, so I just test the inference of secondary antibody in my lab first. I have loaded the same IP sample to three lanes and just add different secondary antibody to see the results. Of course, the contamination of anti-rabbit secondary antibody is very serious, besides the position of heavy chain and light chain, many other bands existed. For the anti-mouse and anti-goat secondary antibody, there are also bands existed on the position of heavy chain. So this problem will bring a lot of trouble for my WB experiments after co-ip. May be the Clean-Blot IP Detection Reagent can help me to solve this problem. Or may be some one can give me some other suggestions.
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Old 12-12-2008, 11:06 AM
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Default Re: problems about western blot after Co-IP

Hi

I haven't used this Clean Blot IP product, so I don't know about that.
Why not just crosslink the antibody to the beads instead. I use BS3 from Pierce.
Roald
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