We transfected HEK cells for a secreted protein and want to confirm the protein is expressed in right MW. Should we collect supernatant for Western?
Not sure we choose the right method. We have tried to detect the cell lysate but was not able to detect a band. I thought the protein got secreated out into the culture medium.
The protein may be secreted but it may be too dilute in the culture medium to be detected. If you are using serum-free medium, then you can use ultrafiltration or trichloroacetic acid precipitation to concentrate the medium so that you can load more protein on the blot.
You can also try immunoprecipitating the medium.
You should also collect the cells, make an extract, and run that on the blot to see if the protein is being made but not excreted.
Finally, it would be beneficial to have a positive control on the blot to make sure your detection method is working.
DEar Cong
if you clone contains a leader sequence for secretion in the media your protein will be secreted in the surnatant and therefore you need to look at this fraction and not to the cell pellet.
I suggest to you to compare surnatant from transfected and non trasfected cells.
You can certanilly load the surnatant in SDS page and compate those in WB but if you use a serum free medium also with standard SDS-page with comassie staining because it you are lucky some proteins are detectable also in SDS if the expression level is high.
On other possibility to avoid WB, if your clone contain an hid tag, is perform a smal scale IMAC (100ul of resin in a Biospin empty coloumn) from the surnatant mixed with imac binding buffer and load the elute in SDS-page. In this way you can concentrate and clean the surnatant and make your protein detectable in SDS-page if the expression level is reasonable.
Because WB, expecially if you use luminescence, is very sensitive and sometimes is difficult from it predict if your productivitiy is really good or not.
I uploaded a short protocool about small imac purification from surnatant of EXPI293 cells, which are improved variant of hek293 in suspension in my research gate profile if you would like to know more detail about it.
good luck
Manuele
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