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how critical is the pH of tbst?


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#1 toejam

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Posted 19 August 2011 - 03:21 AM

hi guys,

I've been working on the amazing world of proteins for the last 2 weeks, specifically learning how to do western blots. At first it went well and had an incredibly strong signal that was even visible in the dark room and 5 s. exposure to the film would "burn" where the positive control (recombinant protein) was present. Then I tried again but the loading of the gels wasn't optimal, so I got contamination on the wells that shouldn't present anything, also got some strange results by what was apparently leaking from one end to the other of the gel.

Until finally yesterday, after overcoming all those inconvenience I loaded a perfect gel, everything was fine (it takes so long to prepare western blots!) but at the end of the day, I couldn't see anything on the film, literally nothing.

A few hours later, a lab mate said that the pH meter was broken and needed to be replaced, so most likely, the tbst didn't have the right pH. Would any of you know how important it is to have the right pH? Do you think that could have been the reason why I didn't see anything at all? Please note, I didn't add the recombinant protein this time to avoid any possible contamination of the samples, but I already knew the size and location of the protein I'm looking for, in case that it appeared.

Anyway, I'm testing now with pbst from tablets instead, since it's my last day of protein work in this lab it's worth to give it a try. Hopefully that will solve the problems.

Thanks in advance.

tj.
"When there's no more room in hell the dead will walk the Earth"

#2 Inmost sun

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Posted 19 August 2011 - 04:22 AM

pH actually has influence on the antibody-epitope binding; I remember when producing my own polyclonals, I routinely tested the binding properties of serum, in 0.2 pH steps of PBS, ranging from pH 7.0 to pH 7.8, to find pH optimum

#3 toejam

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Posted 19 August 2011 - 04:47 AM

hello inmost sun, thank you for your comment. what I was told about the pbst is that I didn't need to pH it, which comes in handy because the pH meter won't be replaced in a couple of days...

so I'm looking forward to the results this afternoon. last chance!

cheers.
"When there's no more room in hell the dead will walk the Earth"




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