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Unspecific binding of multiple antibodies

western blot trouble shooting unspecific

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#1 Ryan Davis

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Posted 02 June 2016 - 01:15 PM

Hello, 

 

I need some help troubleshooting my western blots.  My coworkers and I have been banging our heads up against the wall trying to figure this out...

 

I get specific, strong bands, when probing for my protein of interest.  However, the band is lower than expected.  Expected = ~37 kDa, result = ~18 kDa. 

 

Also, when I try reprobing the membranes for different housekeeping genes (beta tubulin, GAPDH, total ERK, beta actin, and pan actin), I get the same issue:  strong, specific bands that are much lower than expected.  For example, GAPDH expected = ~36 kDa, result = ~14 kDa.  

 

Anyone have any ideas of what could be happening???



#2 bob1

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Posted 03 June 2016 - 07:07 AM

Ok, without many specifics its hard to see what might be the problem - details help in troubleshooting, solutions used (how fresh?), times, washes, temperatures, gel%, blotting conditions, all the good stuff....  This sounds like a bacterial or yeast contamination at one of the steps, I would suspect in either your wash buffer or your blocking buffer or both. Incidentally these bands are non-specific, unless you can eliminate them by pre-incubating the antibody with peptide against the epitope (I'm guessing you haven't tested this). Also check the literature on your protein of interest, it may be that it migrates oddly on SDS-PAGE, this is pretty common, but it is unusual to get a band 50% of the expected size. Also note that some antibodies are just terrible, this may be part of your problem.

 

However, there are a few things you might try: make sure that your block is fresh. If you are using milk powder, make sure that you get a new bag/container and use that, milk powder can grow things even as a powder, and can be contaminated with thing that will grow happily in your buffers once resuspended. Make sure your buffers for washing and blocking are fresh too. Get fresh protein, it could be that freeze/thaw or storage conditions are causing degradation. Ensure that your protease inhibitors are working.

 

Have you tried using detergent such as tween-20 in your block and wash steps? How much salt is in your buffers? How long are you incubating the antibodies for? Are these new antibodies or ones you have had for a while? How are they stored? Can you get new ones in? Do others in the lab get the same result as you when doing things independently (i.e. using their buffers and samples not yours)? If not, get them to repeat using your samples/buffers and compare results.



#3 Ryan Davis

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Posted 03 June 2016 - 08:30 AM

Thanks for your post.  Here is some additional information..

 

Washes:  Wash 3x for 5 minutes with TBST after blocking, primary antibody, and secondary antibody.  The TBST is only a couple weeks old at the max.  

Antibody incubation:  primary is incubated in a 4C fridge overnight.  Secondary is incubated at room temp. for 3 hours.  Both antibodies are diluted in the blocking solution. 

Antibodies: Only a month or two old.  Stored in -20C freezer.  

Blocking:  I either use 5% BSA in TBST or 5% milk in TBST.  The powdered milk package was only opened about a month ago.  The BSA is 6 months old.  I make the blocking solution right before I put it on the membranes.

Gel%: 4 -15% 

Protein samples:  stored in -20C freezer.  They are about 5 months old and they undergo the freeze/thaw cycle probably once or twice a week.  

Another member in my lab is doing some of her own western blots (using different protein samples) and she is getting some unspecific binding but not nearly as much.  Most of the time she gets good data.  

 

I haven't tried pre-incubating the antibody with peptide against the epitope.  I will have to look into that

Perhaps bacterial contamination is the issue.  However, I have used sodium azide to prevent bacterial growth and I still got lower MW bands than expected.  



#4 bob1

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Posted 03 June 2016 - 06:42 PM

Protein samples that are 5 months old and freeze thawed 1-2 times a week will be heavily degraded, most proteins will only really survive a handful of freeze/thaw cycles at best, many only one or two cycles. You should get new samples if possible. If you haven't aliquoted your antibodies and you are freeze/thawing this too, you should get new antibodies also. Antibodies often don't do well with freeze/thaw cycles either. Most antibodies are fine at fridge temperature for a few weeks, though there are (many) exceptions to this rule.

 

The solutions and antibodies should be fine otherwise, though if you are remaking your TBST in the same bottle, it could have carry-over contamination.

 

The secondary incubation could be a bit long, you should be able to get away with 30 min-1 hour, too long will give you high background all over the membrane, not usually in bands, but this can happen too, especially if there is no specific signal. You also don't need to wash between blocking and primary. For non-specific signal you usually want to wash more  times and/or use a more stringent wash (higher salt usually, but also detergent sometimes) between primary and secondary.

 

The peptide incubation step is for validation of your antibody - it is one of the things you can do to prove that your antibody is binding specifically rather than relying on size, as it is well known that many proteins migrate at different sizes than expected in SDS-PAGE. In your case I wouldn't bother with this step yet, unless you can rule out all the other potential problems first.

 

My first step would be to get someone to replicate your work using your samples. If the other person, who normally gets good results has problems with your samples/techniques, then that's where the issues lie. As you state that another in the lab is only sometimes having similar issues, look for common points between your work and theirs, the problem(s) will likely lie there.







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