Hi everyone. Im working with expression of eukaryote gene in bacteria system. I have a question but before that, below is my procedure in the expression.
1. I grow my culture to OD: 06. Upon reach the desired OD, I aliquot 1 ml as uninduced culture. The remaining culture, I induced with IPTG.
2. After induction, I aliquot 1 ml as induced culture.
3. Then, I centrifuge the cell culture, removed the supernant and get the pellet. The pellet, I lyse with sds sample buffer. After lysis, I centrifuge again, transfer the supernatant (soluble fraction) into new tube. But i keep the debris (insoluble fraction).
4. I run both of the supernant (soluble fraction) and debris (insoluble fraction on 12 % sds page.
I have attached my result below:
Lane M: Marker
lane 1: Uninduced culture
Lane 2 - 5: soluble fraction
lane 6 - 9 : insoluble fraction
My question: Since large amount of my protein is present in insoluble fraction, is it indicates that my protein actually were expressed as inclusion bodies?
Hi there,
If the gene product is actually a soluble protein (and therefore not a membrane protein) then yes overexpression in bacteria results certainly in inclusion bodies. To be sure of that protein in pellet should remain insoluble in detergent containing buffer.
Dominique Liger : do you mean that i have to make sure whether my protein is membrane protein or not? and i should directly load the total protein into sds page without separating the debris and supernatant after adding the detergent containing buffer?
If you don't know if the protein is integral, membrane associated or soluble, it is a bit more complicated. The first 2 are not soluble unless you add detergent to your lysis buffer, the third is if properly folded. To check that your protein forms inclusion bodies you have to show that it is still insoluble in the presence of detergent but becomes soluble after massive addition of chaotropic agent like GdmCl (6M) or urea (8M).
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