Sample buffer - Western Blot (DTT)

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Sample buffer - Western Blot (DTT)

Postby newbie17425 » Nov 16 2016 2:34 pm

I have got mixed responses on treating samples after they are in sample buffer and I'm confused which one to follow.

I use sample buffer which consists of Tris-Hcl, Glycerol, Bromophenol blue and SDS. I prepare this and keep it at room temperature and add DTT to this buffer when I have to use it. After I add DTT to this buffer, I freeze it at -20˚C although it is said that DTT is stable at RT.

I add the sample buffer along with DTT at 4:1 ratio to my samples (extracted protein samples) and heat it at 95˚C for 5 min. I aliquot it and keep a working solution at RT. My questions are:

a) Should I freeze the aliquots after heating it or can i keep it at RT?
b) Should I keep the working solution at RT or at 4˚C?

Thanks.
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Re: Sample buffer - Western Blot (DTT)

Postby mdfenko » Nov 16 2016 3:52 pm

a) freeze. briefly reheat when thawed.

b) room temperature, sds will crystallize at 4C.
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Re: Sample buffer - Western Blot (DTT)

Postby newbie17425 » Nov 16 2016 4:04 pm

mdfenko wrote:a) freeze. briefly reheat when thawed.

b) room temperature, sds will crystallize at 4C.


b) How long can I store it at RT? weeks - months?
Do I need to heat it before loading on to gels?

c) If I add DTT to my sample buffer, then should I make aliquots and store at -20˚C?
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Re: Sample buffer - Western Blot (DTT)

Postby mdfenko » Nov 17 2016 7:56 am

b) i wouldn't keep it too long, maybe a couple of days, at room temperature. ideally, you should aliquot one day volumes. the solution should be stable when denatured but why press your luck.

c) yes
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