DsRed fusion protein not glowing - It is there by WB though (Jun/20/2006 )

Hi everyone,

I have created a fusion protein that has an N-terminal Flag tag followed by DsRed and my protein at the C-terminal. I expressed it in 293 cells, but couldn't observe DsRed under fluorescent microscope. My control was fine though. I did lyse the cells and run a WB. I cal see the fusion protein with both Flag Ab and the Ab against my protein. However DsRed Ab won't work (Although I am not sure how good that Ab is). Everything looks in frame by sequencing. I heard that DsRed sometimes may not like the fusion. Has anyone had similar problems, and where you able to get around it. I am currently trying to reclone so that there won’t be a Flag tag at the beginning of the molecule. But in the meantime I am open to suggestions.

Thank you

How long after transfection did u try to c dsred fluorescence ? I have noticed it takes more time than GFP to be visible. Have u done any control dsred transfections to check if u could detect the fluorescence ?

When I tried to detect dsred fluorescence in some other labs microscope (when we didnt have our microscope), the filter was not optimal (although they claimed it was for dsred) and could barely c the fuorescence.

When you did the westerns, was your protein shifted up to an appropriate higher molecular weight you would predict with a DsRed fusion?

Along the lines of what scolix said, I too sometimes can't see fluorescent proteins under the fluorescent microscope when I do indeed have low expression. I can see them with low expression with a flow cytometer though. could this be worth trying?

Are you working with transients or stable cells?

Mountainman

Thank you both scolix and Mountainman for the reply.

I have done the experimetn transiently. Analysed the cells after 24h. There was a MW shift as expected. SO on the WB everything seemed OK. I used a vecotr expressing only DsRed as a control and it was very visible under the scope.

I may try the flow though. It is also possible that DsRed didn't like the particular fusion.

Well I am working on other constructs. Lets see how they will turn out