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Question on loss of signal between samples in western blot

Western Blot SDS PAGE Proteins

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#1 Gradi

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Posted 26 February 2015 - 01:43 PM

Hello all,

 

I have been running western blots to examine protein binding to ribosomes and noticed something interesting. When I run my protein of interest alone in a lane, I am able to successfully detect it by western blot using either my antibody (serum raised against my POI), or a commercial anti-5xHis antibody. However, when I add purified ribosomes to my protein of interest and run this mixture on a PAGE gel and western blot, the signal from my protein of interest either disappears or decreases heavily. I am able to run the ribosomes on their own successfully and have verified their entry into the gel via coomassie stain. This is a new phenomenon which occurred only in the last couple of weeks, as before this I have been able to detect my protein without issue. There is BME in all my loading dye and the dye it self is a 4x concentration used at the correct dilution (to 1x) in the gel sample. 

 

Does any one have any idea why this is happening? 

 

Thanks



#2 mdfenko

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Posted 26 February 2015 - 03:32 PM

what is different the last couple of weeks from before when everything worked well? new solutions? fresh lot of antibody? old lot of antibody?


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#3 Gradi

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Posted 26 February 2015 - 04:41 PM

Same lot of primary (but new aliquot thawed) and secondary. Different preps of protein and ribosomes. My first thought was that it may be something about this new prep of ribosomes. New TBS-T for wash but from same recipe (I mean, buffers do get remade over time). I'm using the same protocol for the WB I used the last time it worked. Do you think blocking over night vs 1hr could change it? Last time I did it and it didn't work I blocked 1hr so i don't think its getting blocked out. Thanks for the response. 



#4 mdfenko

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Posted 27 February 2015 - 07:25 AM

you should not be able to over block unless the block binds to the protein(s) of interest or if the blocking buffer can affect protein binding.

 

did the wbs with overnight blocking work properly?


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#5 Gradi

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Posted 27 February 2015 - 08:45 AM

It has worked fine in the past. 5% milk RT O/N 0.01% NaN3. I wash it after blocking over night to remove the azide (just 2x quick rise with TBS-T and then 1x 5min TBS-T), then move to primary and wash 3x 10min after primary and secondary, so I don't believe the azide is inhibiting the HRP conjugated secondary or anything like that. 


Edited by Gradi, 27 February 2015 - 08:47 AM.






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