| |||||||||
| Login :: Register :: Search forums :: Top Users :: Reagent |
|
Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
|
FLAG westerns
Posted by: margaret hale (IP Hidden, New member, 1)
Date: May 15, 2006 05:00PM
Hi, I'm having a great deal of problems with my FLAG-tagged NUPA10HD expresssion. I have various positive controls which show the FLAG epitope. However, when i try and show FLAG expression in my 293T cells transfected with various vectors containing the FLAG-tagged NUPA10Hd gene, I get very little expression or none. Also, in my PG13 or E86 cells transfected with these vectors, I get an approx. 39-45kDa band when I should be getting a 58kDa band. I have tried the Sigma monoclonal FLAG M2 antibody and a rabbit anti-mouse antibody. I have had these vectors sequenced and I know that my sequence is intact and in frame. When I do real-time PCR, this shows up beautifully. I know that the protein is there. Why can't I show it? Margaret Hale.
Re: FLAG westerns
Posted by: Rosalind324 (IP Hidden, New member, 9)
Date: May 16, 2006 10:58PM
yh....I do the similar experiment before using flag- tag.
first your should confirm that your transfect reagent has no problem.(read its instructions and may find its restriction, or borrow some good reagent from others.) sometime when you have not get enought efficiency in transfection, please try prolong transfecton time if possible.(some transfection reagent can be used up to 16h). So your low expression maybe be solved. In fact ,expression in eukaryotic cell can be shown different as the expression has some relations with the cell lines. if your protein's toxic to your cell? or your some cell itself genome is not stable?
|
We are moving ... Please post to our new community forums
|
|
||||||||