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Immunoprecipitated protein visible on coomassie but not detected by Western


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#1 chimera

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Posted 14 December 2012 - 03:42 AM

Hi everybody,
I am immunoprecipitating my protein with its antibody. I run the eluted proteins onto the gel and could see the immunoprecipited bans on coomassie. But I could not detect the protein by western analysis. What could be the possible explanation for this bizzare phenomena? Please help me. Lots of thanks in advance.

#2 mdfenko

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Posted 14 December 2012 - 07:06 AM

are you sure that the protein of interest transferred well? if it's high molecular weight then it may be difficult to transfer.

that your antibody is in good condition and usable for western blot (check the data sheet and run a positive control)?
talent does what it can
genius does what it must
i do what i get paid to do

#3 Curtis

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Posted 14 December 2012 - 07:13 AM

it's not really bizarre, it happened to us a lot. we just repeated our western. you would also need ponceau to check if the bands were transferred to the membrane..

#4 chimera

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Posted 14 December 2012 - 07:28 AM

I do not doubt the antibody as it detects band for my positive control. Frankly speaking, I did ponseau. I saw heavy chain of the antibody (50 kDa) got transferred but could not understand whether my protein got transferred or not. I thought it might have got masked by the heavy chain (my protein is 40 kDa and is just below antibody heavy chain). May be I should ensure the transfer of my protein to the menbrane and should repeat the western.

#5 Curtis

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Posted 14 December 2012 - 08:27 AM

you sound like you are using same species antibodies




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