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Western blot background issue

western blot background

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5 replies to this topic

#1 jasonrc

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Posted 04 August 2004 - 11:58 AM

Hi,

I have been having a problem with Western blot for the Dopamine receptor 2 (D2). The background comes out black and the appropriate bands come out clear. The rainbow contains both clear and black bands and there are some dark (darker than the background) bands present in the sample lane. The D2 bands always come out strikingly clear. The problem is specific to this antibody; our lab has had no problems with the secondary antibody. The manufacturer insists that it is a development problem. However, our lab has also not had any problems with development of other WBs. Our initial thought was that the signal was too high. We have cut the sample amount in half and titrated from 1:800 to 1:10000 primary antibody concentrations. The manufacturer reccomends 1:800 and publications used 1:400. The result is the same, except for the 1:10000, where a strong background is still evident, but the white bands are faint. The 1:5000 did have a lighter background, but the bands were still very clear. Three different blocks have also been used (goat milk and serum and cow milk). I also thought that excess second antibody may be an issue. I rewashed the membrane for several hours and redeveloped with no decrease in background. I appreciate any help that can be offered.

Thanks,

Jason
University of Michigan

#2 Proteinjunkie

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Posted 05 August 2004 - 01:52 PM

How long are you exposing the blot for? You might want to cut it down to just seconds...

#3 jasonrc

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Posted 06 August 2004 - 04:56 AM

I have exposed for as little as 1 sec. The problem is the same, only a little fainter. I may have some idea about the problem. Because of reactivity problems, only goat's milk or serum can be used as a block. BSA is known to give extremely high background. We always dilute our antibodies in a 2.5% BSA solution. I did several dot blots (protein directly onto the membrane, without gel separation) and ommitted BSA from the primary. The 1:10,000 looks pretty good, whereas the bands were still reversed when I did the standard Western at this dilution (containing BSA). Could someone tell me if this is possible: the antibody binds strongly to the antigen, but also reacts with BSA in the solution. Subesequently, the secondary antibdy is blocked from binding to the primary complex, but creates a high background everywhere else. This is the only way I can rationalize the presence of clear bands, with high background and some dark bands. I think I will take a few days off from Westerns and try again without BSA (perhaps using goat serum).

#4 jasonrc

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Posted 17 August 2004 - 03:50 AM

Omitted BSA from both primary and secondary dilution; problem solved!

#5 lepetitprince

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Posted 19 July 2012 - 10:52 PM

For the sake of keeping the Dopamine story together, I am posting here:

My question is about antibodies for DRD1 (Dopamine Receptor D1).

Several biosciences companies mention the molecular weight of DRD1 as 49 kDa (Abcam - http://www.abcam.com...dy-ab20066.html) to 75 kDa (Santa Cruz - http://datasheets.scbt.com/sc-1434.pdf). Recent papers confidently believe that DRD1 is at or around 49 kDa (http://www.ncbi.nlm....pubmed/22336227).

Abcam's datasheet at least honestly report thus: "Observed band size : 48 kDa, Additional bands at : 45 kDa,70 kDa,75 kDa. We are unsure as to the identity of these extra bands.

I know that Dopamine receptor antibodies are notorious for poor sensitivity and specificity.

Any help as to what these other bands are and why different companies are confidently claiming the Mol Wt of DRD1 variously as 49 or 75 kDa shall be greatly appreciated.

#6 bob1

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Posted 19 July 2012 - 11:59 PM

I have had dodgy antibodies from both Abcam and Santa Cruz (though also good ones from both too), and the data sheets in both cases were indicating the wrong size for the protein, despite referencing papers that said the correct size. If papers refer to the size as 49 kDa and this is +/- 10 kDa of the predicted size from amino acid sequence, then you can be reasonably confident that 49 kDa is correct.

Different bands in an antibody are sometimes different forms (e.g. dimers) of the target protein, but more commonly are not related to the target protein at all and there is no way to tell what they are other than to protein sequence them.





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