I'm trying to express membrane protein gene in pACMV-tetO Flag taged one. I did a transient expression in HeK293 Gent- but when I did a western blotting (without purification) only the cell lysate I didn't get any signal on the membrane. I tried it twice fro different constructs so it's normal for transient expression and I have to wait for stable cell line or no I have to see expression? I have no GFP in the constructs
Hi Marwa
You need to double check the sequence of your plasmid and ensure the correct frame of amino acid. In fact you had to detect the band of interest! I recommend that you use SDS-PAGE and see if you could detect the band. Then you can further follow the troubleshooting. Coz WB is a long process and there are lots of chance for the problem to occur. If you detect it by SDS-PAGE then you should check you antibodies.
Hi Amin,
Actually yes I did a sequencing and it's on the right frame as already designed. Usually I'm staining SDS-PAGE after blotting to determine the efficiency of blotting and usually I've bands for sure you know the blotting isn't 100%. But remember I can't detect my protein on SDS cause I'm not purifying it and using the whole cell lysate so I have to detect it with the western and I\m working with a positive control for FLAG tag and it\s easily detected but this is coming from stable cell line not transient one!
So mainly my question is that in case I\m using the whole cell lysate should I've to detect the expression after 72h for transient expression of the above mentioned construct or it's difficult to see it?
Hi,
so far HEK293T proliferate quite fast, I would choose also 24hr and 48 hr to detect the Protein. Is it possible for you to check the Expression on mRNA Level? And can you maybe stain your cells directly? It could be also possible that your extraction buffer is not suitable for Membran Proteins. And a question regarding the Plasmid, is the Expression under a tetracyclin Promotor? your wrote tetO. Sometime you have also tetracylin in the FCS.
Hi Urda,
it's not possible to check the expression on mRNA level but I don't understand how to stain the cells? And yes it has a tetracycline induction and I\m inducing it
Why it is not possible to check the mRNA expression? when you induce the Expression of the gene, there should be mRNA. Especially with tet-inducing it's more or less important that you can Switch on and of the expression. For the stainig, you can seed you cells for example on coverslides, fix them and stain them with your antibody directly - immunfluorescence. Or you trypsinize you cells, fix them, stain and Analyse by FACS.
it's not possible cause I don't have a real time primers for it. Regarding to the staining of the cells I'm working with FLAG taged protein this is convenient with FLAG tags ?
Recent work showing FLAG epitope-tagged membrane protein transient expression in HEK 293 cells. WB, IP and in-cell IF detection methods used. This should give you some ideas:
http://onlinelibrary.wiley.com/doi/10.1002/jcb.25574/full
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