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Most annoying Co-IP ever! Halp!

Co-IP

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#1 Cinisia

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Posted 27 September 2012 - 03:31 PM

Looking for any kind of thoughts/ideas/wakeup call/donation/etc to get over this IP! Thanks in advance!


Soooo I've got a project passed down to me from a previous undergrad in the lab. The project is trying to verify interaction between protein A and B by Co-IP & Western blotting. 2 out of 4 pulldown & mass spec had shown that they interact.

The plan is to transiently transfect the two proteins in a drosophila cell line and do a Co-IP because the endogenous level of those proteins are very low. However I have made a few attempts with very disappointing results. Aside from unstable protein expression (which I'm working on right now) there are a few more problems. So far I've only succeeded once and the blot was of too poor a quality...

1. There are no antibodies available for those proteins... So both proteins have to be tagged *shrug*. I'm also considering cross-linking with PFA to capture the interation because past experiments (the previous student's lab book) suggest that IP without cross-linking had been mostly failing. My question is, with all that cross-linking and tagging does whatever I'll be able to find (or not) have any biological significance?

2. The second problem... I've been using the lysis buffer containing 1x Tris, 1mM EDTA, 1% NP-40, 2mM Na3VO4 and one of Roche's protease inhibitor cocktail tablets. But upon lysis there's this white sticky substance in the lysate that filtering can't remove. It has been quite bothersome when it comes to loading my input samples. Any suggestions as to why or how to avoid it?

3. Background... and lots of it. On my Western there are consistently three background bands which just happen to overlap one of my proteins. They are at 80 ~140kDa. I suspect the reason to be that I've been using antibodies from the same species to pulldown the protein and to probe the Western blot. I have tried increasing the salt concentration in my wash buffer and even washing with PBST, neither worked. Any suggestion on this matter would be great.

Again thanks for taking the time to read all this stuff... I would greatly, greatly appreciate any suggestions!

Thanks!!!

#2 bob1

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Posted 27 September 2012 - 08:06 PM

1) your thoughts are good ones - I would be a little doubtful too. Preferably the interaction would be determined in 2 or more ways - confocal microscopy for co-localization and in vitro transcription are commonly used for confirmations.

2)Hopefully there's some salt in the lysis buffer too - preferably in the range of 100 -300 mM. The tris needs to be about pH7-7.5. The white substance is probably DNA or perhaps something specific to the insect cells such as chitin. These can be very hard to remove, aggressive centrifugation for extended periods of time can help. DNases such as Benzonase might help. You could also try lysing at a lower number of cells per volume.

3)The heavy and lght chains from antibodies used in the IP can obscure bands, but these are normally around 55 and 20 kDa respectively. If you think this is the problem you can get protein-G conjugated with your detection system which will stop the detection of the heavy and light chains. It is more likely that you have some non-specific binding of your antibodies to something in the lysate. What do the control lysate westerns look like (westerns of the input lysate only - not IPed)?

#3 Cinisia

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Posted 28 September 2012 - 09:12 AM

1) your thoughts are good ones - I would be a little doubtful too. Preferably the interaction would be determined in 2 or more ways - confocal microscopy for co-localization and in vitro transcription are commonly used for confirmations.

2)Hopefully there's some salt in the lysis buffer too - preferably in the range of 100 -300 mM. The tris needs to be about pH7-7.5. The white substance is probably DNA or perhaps something specific to the insect cells such as chitin. These can be very hard to remove, aggressive centrifugation for extended periods of time can help. DNases such as Benzonase might help. You could also try lysing at a lower number of cells per volume.

3)The heavy and lght chains from antibodies used in the IP can obscure bands, but these are normally around 55 and 20 kDa respectively. If you think this is the problem you can get protein-G conjugated with your detection system which will stop the detection of the heavy and light chains. It is more likely that you have some non-specific binding of your antibodies to something in the lysate. What do the control lysate westerns look like (westerns of the input lysate only - not IPed)?


Thank you so much for replying! For my lysis buffer the salt concentration is 100mM. The strange thing is when I use RIPA buffer to lyse cells there was no white substance. It only shows up when I use the IP lysis buffer. As for the background bands they are only in the IPed sample. Input samples are clean as they can be.





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