I am trying to express a novel GlcNAcT enzyme in BL21(DE3). I have successfully cloned it in pET28a vector and confirmed it through sequencing. I tried following previous protocols for GlcNAcT expression but did not see any protein expression. Induced and uninduced samples both are looking the same in SDS-PAGE. What might be the cause, and how can I resolve this situation?
Might be a codon usage issue. Which organism is the cDNA from?
Hi there,
I guess you tagged the protein with 6His. Did you try WB for revelation? No difference in Commassie staining patterns doesn't mean no expression (it could be low expression). Making the difference between no and low expression is quite important: if no expression there might be a construct issue, if low expression then optimization may be considered.
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