Help:protein phosphorylation

Tatyana Nikitina TN3532 at TN3532.spb.edu
Fri Nov 13 11:15:52 EST 1998


Vladimir Svetlov wrote:
>
>What led you to believe that this is the case (were the phosphorylated
>subunits labelled with P32/33, were they assayed with anti-phosphoY/S/T
>antibodies etc.)?


The subunits were labelled with P32.

>I believe that the
>problem you are experiencing is not with the SDS-PAGE of phosphorylated
>subunits per se but either with using too high percentage of the gel (they
>do tend to run slower than non-phosphorylated ones at least in some gel
>systems) or with protein agrregation.

We also think that our problem is with protein aggregation but we can not
reveal the nature of this aggregation. It seems that only phosphorylated
subunits aggregate because when we stained the gel after autoradiography
with silver we could see that nonlabelled subunits had been separated.

>Make sure you have fresh sample buffer with plenty of BME.

We use sample buffer with 2% BME. May be we should use higher
concentration of BME?

<You can also try loading your proteins in 6 M
>urea (unlike GuaHCl it does not precipitate and does not significantly
>affect the separation) - this should pull most of your subunits apart. I'd
>try to use 4-20% or 6% gel with Tris-glycine or phosphate buffer while
>making sure that all the components/operations affecting denaturation of
>the sample (heating, BME, and SDS) have been added/carried out.
>Good luck.


We've never used phosphate buffer. Are there any differences between the
use of phosphate and tris-glycine buffer in SDS-PAGE? And what is the
concentration of phosphate buffer you use?

Thank you for your message.

Tatyana Nikitina
St-Petersburg, Russia



>V.
>

>Vladimir Svetlov, PhD
>McArdle Lab for Cancer Res.
>UW-Madison
>1400 University Ave.
>Madison, WI 53706








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