interested protein shows in every sucrose fraction! help! - (May/21/2008 )

I used 5% to 25% sucrose gradient to seperate nuclear extract, and do western to see where is the protein complex?
And I see my interested protein shows almost every fraction!!
anyone can give me some advice???????
should I try to use high concentration of sucrose? ie 10%-50%? or whatelse could I try?

I really do not have many clues on it!!!
Thanks million.

-cathy-

QUOTE (cathy @ May 21 2008, 09:16 AM)

I used 5% to 25% sucrose gradient to seperate nuclear extract, and do western to see where is the protein complex?
And I see my interested protein shows almost every fraction!!

anyone can give me some advice???????
should I try to use high concentration of sucrose? ie 10%-50%? or whatelse could I try?

I really do not have many clues on it!!!

Thanks million.

the sucrose gradient you use is to separate subcellular fractions but not normal sized proteins; better use gel filtration to separate proteins

-The Bearer-

QUOTE (The Bearer @ May 21 2008, 11:41 AM)

QUOTE (cathy @ May 21 2008, 09:16 AM)

I used 5% to 25% sucrose gradient to seperate nuclear extract, and do western to see where is the protein complex?
And I see my interested protein shows almost every fraction!!

anyone can give me some advice???????
should I try to use high concentration of sucrose? ie 10%-50%? or whatelse could I try?

I really do not have many clues on it!!!

Thanks million.

the sucrose gradient you use is to separate subcellular fractions but not normal sized proteins; better use gel filtration to separate proteins

I did not understand well. lots papers people use gradient to seperate protein complex and showed that proteins interacted with each ohter showed in the same fraction. why not I can not use it?

-cathyli-

1. Some technical issues.

2. Your protein may be forming many complexes of different sizes. It is not necessary that your protein binds to only one group of proteins exclusively.

For example: Your protein is X,

complex A-B-X 300 kd
Complex C-D-B-E-X 500 kd
Complex M-N-T-U-V-A-Y-X 2000 kd

-cellcounter-