SDS-PAGE shows two bands ?

classic Classic list List threaded Threaded
2 messages Options
Reply | Threaded
Open this post in threaded view
|

SDS-PAGE shows two bands ?

Stephen McMahon
Hi,

 

I have purified a protein of unknown sequence that runs as a single peak on
gel filtration.  An analysis on SDS-PAGE shows two bands.  Before staining
the gel (coomassie) the upper of the two bands is already clearly visible
(~24kDa).  The lower band only becomes visible after staining (12kDa).  My
feeling is the two bands represent the same protein and the upper band has
something covalently attached (some sort of dye) such that the protein runs
erratically on SDS-PAGE and allows it to be visualized without staining.

Does anyone have any experience of this or have any idea what is attached to
the protein?

 

Many Thanks,

Stephen

 

_______________________________________________
Proteins mailing list
[hidden email]
http://www.bio.net/biomail/listinfo/proteins
Reply | Threaded
Open this post in threaded view
|

Re: SDS-PAGE shows two bands ?

allisonh
Stephen McMahon wrote:

> Hi,
>
>  
>
> I have purified a protein of unknown sequence that runs as a single peak on
> gel filtration.  An analysis on SDS-PAGE shows two bands.  Before staining
> the gel (coomassie) the upper of the two bands is already clearly visible
> (~24kDa).  The lower band only becomes visible after staining (12kDa).  My
> feeling is the two bands represent the same protein and the upper band has
> something covalently attached (some sort of dye) such that the protein runs
> erratically on SDS-PAGE and allows it to be visualized without staining.

What size does your protein elute at from the gel filtration column?  If
it is around 36K (or other combination of 12K and 24K) then run a
non-reducing SDS-PAGE -  your protein may have subunits held together by
disulphide bonds.

Does the protein peak from the gel filtration column have the same
colour as the band on the gel?

Allison
_______________________________________________
Proteins mailing list
[hidden email]
http://www.bio.net/biomail/listinfo/proteins