I am using pIEX/Bac-3 vector for expression of my protein into sf 21 cells. After transfect ion how can I conform the expression of my gene. Any PCR method is available for the detection of target gene expression. In my vector no marker available for detection.
1) SDS PAGE and look for band at right size in whole cell lysate. The band should be much thicker/darker than the others.
2) Is there a biochemical assay to detect your protein/protein's activity? Take whole cell lysate and run it in the biochemical assay and compare it to a non-transfected control.
Hello,
What is your transfected protein? You can do immunoprecipitation or Western blot analysis with whole cell lysate (if you have the antibody against your protein of interest).
You also can check and quantitate the expression of the gene of interest by Real time qPCR.
You can do immunofluorescence studies with the transfected cells to see the localization of the protein of interest (you need to suitable antibody and fluorescence-tagged secondary antibody).
Your recombinant protein should have an N-terminal 10 His-tag or a C-terminal Strep tag when you use this vector. The easier way to confirm the expression is to do a western blotting using either anti-his or anti strep antibody which are commercial available.
western blotting using the antibodies against the affinity tag is the easiest way.
I recommend to firstly to design primers for PCR, the one surrounding the transcription start site TSS (upstream of your insert, and the reverse is downstream of TSS) , then you can check by PCR, if so, this is mean your cassette is already inside the cells but also not sufficient to indicate it is expressed or not, then you can go to the western blot using anti tag if your insert has it , if not you can detect using specific antibody against your target protein if the cells does not express endogenous one , and this by western or immunofluorescence
hopes it can help
Western blot is definitely a way to go. You can do it with both antibody against your protein and antibody against the tag, if the recombinant protein is tagged
Western Blotting. If Protein yield is the problem ? - try Chemiluminescence based Immunoblotting.
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