chemiluminesence on western blot

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chemiluminesence on western blot

Postby Sophs012 on July 13th, 2015, 6:01 am 

Hi All,

I've just been looking back at my lab books, and noticed the volume of chemiluminese reagant can vary (although the membrane size can vary and this is why the volume varied) - one or two had a bit more on the membranes proportionally to the size (so a ~1/4 membrane had 400 microlitres when technically id use that much on half a membrane) - how much would this effect my results?

Sophie
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Re: chemiluminesence on western blot

Postby SciameriKen on July 13th, 2015, 8:13 am 

400 ul seems like it would be difficult to spread evenly on the membrane. Assuming equal spreading the only thing that chemi agent would be affecting is how bright the bands are. If you are comparing between samples on the same gel it should not matter as there is no difference between their relative luminescence, however if you are comparing samples between two separate gels then there might be an issue with that.
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Re: chemiluminesence on western blot

Postby Sophs012 on July 13th, 2015, 9:04 am 

Hi

I'm comparing samples within one repeat on the same membrane, but in a way comparing the trend between repeats and averaging out - is this something to consider? I also compare the protein to beta actin, again its all proportional within the same conditions

Sophie
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Re: chemiluminesence on western blot

Postby SciameriKen on July 13th, 2015, 9:54 am 

Sophs012 » Mon Jul 13, 2015 1:04 pm wrote:Hi

I'm comparing samples within one repeat on the same membrane, but in a way comparing the trend between repeats and averaging out - is this something to consider? I also compare the protein to beta actin, again its all proportional within the same conditions

Sophie


I don't think the luminescence really varies that much to really matter in this case - but assuming it does, do you run your actin at the same time as your protein of interest or do you reblot? How much of a difference in chemi agent are we talking about?
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Re: chemiluminesence on western blot

Postby BioWizard on July 13th, 2015, 7:22 pm 

As long as you have enough solution to cover the membrane, and your are normalizing to a loading control (I guess you're using actin), you shouldn't have a problem.
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Re: chemiluminesence on western blot

Postby Palladium106 on August 26th, 2015, 6:47 am 

Volume is more of a voodoo thing in this case. As long as you have enough. Cost saving May be a consideration for some? If you are using a rocker you should be ok with a small volume.
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