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Some bands of similar intensity aren't seen in short exposure but are seen i

western blot non-specific binding help

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#1 leighann36



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Posted 15 February 2015 - 06:12 AM

Hello all!

I performed my second ever Western blot and I am somewhat confused about my results. I was checking for the expression of a fungal effector protein in barley leaf extracts. In the short exposure, I saw much less (understandably) but the curious thing was that some neighbouring bands, of similar intensity, were not observed in the short exposure but were seen in the long exposure.


Short exposure (3 minutes):

short expo.jpg


Long exposure (30 minutes):

B19 (2).jpg


Any reason why this could be so? 


Also, I loaded twice as much protein than in my first Western and I observed much more non-specific binding. Is this normal? 


Many thanks,



#2 mdfenko


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Posted 15 February 2015 - 01:29 PM

those bands may appear to be of equal intensity because they may have saturated the film during the longer exposure.


the increase in non-specific binding with increased protein is sort of normal. more of the protein may break the threshold of detection. 

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