We express different constructs of cadherin domains from desmoglein-2 (DSG-2) carrying either one cadh domain, two or four cadh domains. All the constructs have a His-tag in C-ter and are expressed in HEK293 cells by transfection. We are unable to see the protein (cell or supernatant) by western blot using anti-His antibody. However we have checked by immunofluorescence using anti-his labelled with a fluorescent dye and the protein is really expressed in the cytoplasm. Any of you have already encountered this kind of problem with cadherin expression? Any idea that would help?
The amount of protein that is produced in the transfected cell lines is too low that can be detected, the only option is to concentrate the supernatant using centricon and check for expression, further you can develop the blot by chemiluminescence as the sensitivity is high and low amounts can be detected.
My impression is that his-tag antibodies are somehow picky: when we test a range of different anti-His antibodies for immunoblotting, we're usually happpy if one of them gives a nice blot for any given protein...
Some antibodies work well under native conditions (IF), then fail in denatured applications (western blot). Find another few His-tagged proteins - express them, make lysates. These will tell you if you antibody works for westerns. Alternatively- look for the antibody you use in publications for western blotting- can there get it to work? Finally- you may need multiple tags- His is pretty small from memory....
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