For this experiment I am using two lysates. First one has phosphatase activity and other one does not have the phosphatase activity. I wanted to see the affect of phosphorylation in JAB1. For this experiment can I use lysate directly without purifying the protein? My plan is to run phos-tag SDS-PAGE and immunoblotting for visualization.
You can use Lysate directly but i guess without purification of protein the results will not be very clear.
your question not clear for me. however, i ll answer as far as i understnd. if you want to detect the phospho activity for any protein i would add phosphtase to your lysate to elimate any possible phosphoraltion that could heppen for any reason.
Hi
You can definitely use total lysate prepared in medium devoid of phosphatase enzymes to prevent degradation of your protein of interest. However, if you cannot purify the protein completely, if is advisable to use monoclonal antibody to recognize the tag by SDS PAGE.
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