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Open Forum on Methodology

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15 December 2000

Joe Brozinick

We have been doing GSK3B IP kinase assays from intact muscle tissue lysates. These lysates are made from muscles that have been exposed to insulin either by in vivo injection of insulin (30 U/kg BW for 10 min) or by in vitro incubation with 133 nM insulin for 20 min. We have been using the GSK3B monoclonal antibody from Transduction labs. The assay was working very well, but the in the last several months we have been detecting an increase in activity with insulin, rather than a decrease. We have tried different assay buffers, different homogenization buffers and different substrate peptides (CREB,EIF2, and GS specific) but have gotten the same results. Does anyone have any suggestions?

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