I am going to start pull down experiment to check the protein protein interaction initially confirmed through yeast two hybrid screening. I have cloned the gene of interest in PCOLD-GST vector. I want to check first the efficiency of protein production and to find out whether the protein is soluble or insoluble and its efficiency with induction with adding IPTG. Please help me to find out proper methodology to perform these initial steps.
Thanks.
Hi there,
One way to estimate protein solubility is to compare the protein pattern of a total lysate sample (ie. right after cell breakage) with the one from the same lysate which has been clarified by centrifugation (20 minutes at 20 000g). As a control the same approach has to be taken for the uninduced culture cells. If the protein is present with the same band intensity (provided the loaded volume is the same for both) in the 2 samples, your protein is soluble. If it is present only in the total lysate sample, it is completely insoluble. If band intensity decreases from total lysate to clarified lysate then the protein is partially soluble.
Generally, if expression level is good, Comassie staining is enough to spot the protein. If not, WB may be considered.
Dear Dr. Dominique Liger, Thanks for the answer. I am very clear about the overall method to follow. I am little confused with how to prepare total lysate sample and its further processing for protein sample preparation. It will be nice if you could elaborate the detailed protocol followed in general. Thank you very much.
Well, cultured cells have to be harvested and resuspended into lysis buffer prior to lysis (I usually use sonication) then an aliquot of the lysate (which is actually a suspension of cell débris) is mixed with SDS/PAGE loading buffer then the rest of the lysate is centrifuged and finally the same aliquot (in volume) of the cleared supernatant is mixed with SDS/PAGE loading buffer.
Now everything is very clear to me. Thanks for your kind responce.
http://onlinelibrary.wiley.com/doi/10.1002/bab.24/full
This publication may be of some assistance in general methods about protein expression
I am having strange results with my Western blot results. My protein of interest is suppose to increase after treatment. Infact it has decreased compared to the control. Does this mean the proteins have went into insoluble fraction or there is something else happening? Some studies have shown that my protein of interest sometimes do go to insoluble fraction. Can someone help me explain this? Also if you have a detailed protocol to isolate insoluble fraction of protein, that would be a great help. Thank you.
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