I am trying to express a GST tagged protein in BL21. The size of the protein is 38kDa so the expected band is supposed to be around 64kDa (plus 26 for GST). However, the band I got was just above 25kDa as compared to the ladder, suggesting it is a band for the GST. I carried out expression for another protein along side as a control and the control shows the expected band. Please does anyone know what could be the reason for this and how I can go about solving this problem?
Olayinka Akinpelu , there might be two possibilities
1. Either your protein is expressed in inclusion bodies (insoluble fraction)
2. or your gene was not incorporated during cloning.
Try confirm your clone by done restriction digestion first or sequencing, if you did, check also the inclusion bodies if your protein is there.
Sibusiso Maseko I have done restrictions and it confirmed the clone was there, although I am still waiting for sequencing result. But then, if the protein was expressed in inclusion bodies, why is band for GST showing? Isn't the GST suppose to be with the protein in inclusion bodies?
You always get a small amount of GST that is not fused with to your protein expressed all the time, especially if you have inclusion bodies.
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