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black blot images on AI600 imager, .tif shows faint bands - (Nov/03/2015 )



I recently started to do western blots and I'm currently testing several antibodies against MAPKs that none im my lab used before, so I have to find proper conditions to use them for my samples.

Some antibodies gave nice blots, others took a bit tweaking with blocking and primary antibody conditions,but with others I have that problem that when I take photo (using Amersham AI600 imager) i get black or blotchy photo. That is photo captured in mode with colorimetric marker. Whereas, .tif images of the same blot (it consists of 2 layers, picture of detected proteins and of standard), sometimes have only faint bands, without black blotches, and sometimes just grey blotches.

I played with conditions, tried changing blocking buffer 5% BSA to 5% skimmed milk, for 1h or overnight, antibodies in 1% BSA or skimmed milk overnight at 4C or 2h at RT. Even tried primary antibody diluted without blocking agens or in 5% BSA or skimmed milk. Tried diluting or concentrating primary antibody and diluting secondary but without success. I also tried different concentrations of protein, 20-40 ug per well.

I even briefly thought it is some problem with capturing of images or chemiluminescence detection reagens (which we make ourselves in lab), but then I wouldn't get nice photos for any of the antibodies.

Does anyone have a suggestion what else could I do to solve my problem?

Images are attached.



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-Andrea H-

if all others besides this one work cleanly then, assuming that you use the same secondary antibody as those that worked, the problem is with your primary antibody (i realize that i'm only stating the obvious but bear with me).


what about this antibody is different from the other primaries?


is it specific for phosphorylated protein? if so, then you should not block with milk, casein is highly phosphorylated.


(if you're using a biotinylated detection system then you should also avoid blocking with milk)


have you tried blending in normal serum from the species in which the secondary antibody is produced with your blocking and antibody solutions? this would preabsorb any primary that will react with that species proteins. it's especially useful if you use a polyclonal antibody.


if you're using a monoclonal antibody, are you sure that the epitope against which it is produced is not found in other proteins?


you can find a useful handbook on western blotting at this ge lifesciences webpage (under the "protein analysis" tab):


i'm also attaching a western blot troubleshooting guide from boster and the protein blotting handbook from millipore.

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