I'm doing in-gel kinase assay.
The substrate was mixed to resolving gel to let it be co-polymerized within the SDS gel and run at 12.5 mA.
I worried that the substrate will also run during electrophoresis. So, after SDS-PAGE running (around 2.5h, the dye still in gel), the gel was performed western blot. However, I only got the band of protein substrate around the end of gel. Substrate run, instead of in gel.
The substrate for the in-gel kinase assay is a protein so you do expect it to migrate through the gel during electrophoresis. This is expected and not a problem.
here's a paper with a good summary of the technique:
Article Assessing Kinase Activity in Plants with In-Gel Kinase Assays
I wonder if you need to use gel with reduced SDS or no SDS (native gel) to allow kinase to be enzymatically active on the substrate that is present within the gel. Further, in this case "separation" would depend on net charge on the kinase and substrate (and not on the size of the substrate).
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