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No bands can be seen in my co-IP


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#1 blixx

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Posted 25 October 2014 - 08:55 PM

Hi everyone

 

I did a co-IP last week but couldn't see any bands when I developed my membrane. This is a repeat co-IP and it worked perfectly well the first time. Not sure exactly where I went wrong but I think this time around I added 300uL of lysis buffer to 15cm dish (same as last time), scraped the cells and then ended up with 600uL of cells+lysis buffer whereas last time I had around 350uL. Perhaps I haven't removed all the PBS when I washed the plates? However, I IP'd only 300uL of the lysate and stored the rest as a pre-IP.

 

Could it be that because I only used half my lysate that I couldn't see any bands? Can this be fixed by re-IPing my stored lysate, use a smaller vol of beads/loading buffer and then add that to my previous IP to push the protein concentration up?

 

Please advise.

 

Cheers!



#2 miST32

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Posted 26 October 2014 - 12:56 PM

Hi blixx,

Can you see your target antigen?  It might be possible that your co-purified protein is present in exceedingly low quantities if you halved your input, but I'd still expect your IP to work.  If you see no band for the target antigen, then the IP itself failed and you shouldn't expect any co-IP.

I find it helps to always normalize my IPs by using equal concentrations and volumes.  For example, you could always IP from 500uL lysate solution at 1mg/ml concentration - or whatever conditions are necessary for preserving the interaction.  I just find it helps standardize the process, and makes loading the input (pre-IP) facile, since it's always a simple % volume of the lysate used for the IP.



#3 miST32

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Posted 26 October 2014 - 12:59 PM

You might be able to visualize the IP by simply staining the membrane with ponceau.  You should see strong IgG bands and if the IP is robust, you should also see your IP target protein at an appopriate MW.  This isn't always the case if the IP is weak, but it would be a quick way to test.  Compare to your old membrane if you have it saved, and see if both yielded the same IP quality (don't worry about the co-IP at this step - the IP has to work first).

 



#4 blixx

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Posted 27 October 2014 - 02:05 PM

You might be able to visualize the IP by simply staining the membrane with ponceau.  You should see strong IgG bands and if the IP is robust, you should also see your IP target protein at an appopriate MW.  This isn't always the case if the IP is weak, but it would be a quick way to test.  Compare to your old membrane if you have it saved, and see if both yielded the same IP quality (don't worry about the co-IP at this step - the IP has to work first).

 

 

 

Thanks miST32. The ponceau test did not work. I'll re-run the co-IP again this week.

 

Cheers!






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