pH neutralisation of samples for SDS-PAGE

Nick Theodorakis nick_theodorakis at hotmail.com
Thu Mar 30 12:42:01 EST 2006


Amanda Gillespie wrote:
> I have neutrophil homogenate samples, which I am treating with a glycanase
> to deglycosylate the protein I am interested in, to be followed by reverse
> zymography to test the activity of this protein.
>
> Any suggestions/opinions/comments on how to neutralise the pH after
> deglycosylation, as the buffer required for this is very low -
> 5.0citrate-phosphate, which is considerably lower than the optimal pH
> for my
> protein, and inteferes with the activity shown on the reverse zymogram...
>
> I tried just adding normal non-reducing treatment buffer to my samples after
> deglycosylation, hoping that would be enough to adjust the pH, but
> apparently, it isn't :(

Assuming you are using bromophenol blue as the indicator dye-- make a
small stock of unbuffered saturated Tris base. Titrate in small amounts
(e.g., 1 or 2 microliters or so) of base into your sample until the dye
turns from yellow/orange back to blue.

Nick

--
Nick Theodorakis
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