Some Minor Questions Re SDS PAGE :

Neal Melvin nrmelvin at ucalgary.ca
Sun May 7 16:07:52 EST 2000



Mehdi Alimadadi wrote:

> Dear Scientists,
>
> I need answers to the following questions:
>
> 1.  Should the protein standards be diluted with loading buffer before
> loading or
>      it is not needed?

Yep... they need to be diluted in a denaturing solution (your loading buffer)
that contains a disulphide bond breaker (beta-mercatoethanol or
dithiothreitol) and SDS

>
>
> 2.  Do the protein standards also need to be boiled before loading?

Yep

>
>
> 3.  Once boiled for 5 min with loading buffer, the protein stock samples
> could be
>      stored in -20 but do they need to be boiled again each time before
> loading?

Yep

>
>
> 4.  When I have far different concentrations of proteins, do I need to
> precipitate and dilute again in order to have approximately similar
> concentrations within equal total volume, in order to have a perfect run?

I would suggest using spectrophotometry to quantify the protein in your
samples, then diluting them all down to the same concentration... then from
here, take equivalent volumes from different sources (which will now contain
the same amount of protein) and load on your gel.

Neal





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