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Ab smear obscures signal on IP-western

Ab smear obscures signal on IP-western - Protocols and Methods Forum

Ab smear obscures signal on IP-western - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 08-03-2009, 06:48 PM
DK
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Default Ab smear obscures signal on IP-western



In article <[Only registered users see links. ].net> , Lionel Brooks 3rd <[Only registered users see links. ]> wrote:

When you denature without reducing conditions, you potentially expose
all those buried cysteines, which are now free to form S-S bridges. When
there are many proteins in the sample, this leads to a mess (particularly
if the samples are heated). The solution is simple: add ~ 50 mM iodoacetamide
to your SDS page loading buffer (it's soluble in water up to ~ 0.6M).
I do it all the time. Acetamide is not terribly stable and is light-sensitive,
so use it fresh. NEM is an alternative but IAA reacts faster. This will also
"neutralize" all of your DTT in the sample. (Native, non-denatured IgG is
normally not reduced by 1 mM DTT, so it's unlikely that your 20 kDa ~ light
chains are there before you mix your sample with loading buffer.

DK



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Old 08-05-2009, 04:29 AM
DK
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Default Ab smear obscures signal on IP-western

In article <[Only registered users see links. ].rossu.loc>, "Dr Engelbert Buxbaum" <[Only registered users see links. ]> wrote:

Engelbert,

Didn't you mix up something? The OP stipulated "must be performed
under non-reducing conditions". TCEP is a strong and stable reducer.
It surely will prevent spurious S-S formation but will also break
preexisting ones.

DK
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