Hello,
I currently have sliced and punched mouse brain tissue using the cryostat and I wish to analyze it for the concentrations of different dopamine receptors. We initially desired to use Western Blot analysis, however, the protein extraction (via BCA assay) determined that the total volume exceeds the maximum well capacity of 15 ug. We are now looking into different alternatives to the Western Blot analysis, like the use of Polyacrylamide gels followed by nanodrop technique.
Does anyone have any experience on the polyacrylamide gel method of protein purification and measurement, specifically for punched samples? Perhaps on the feasibility of method or useful tips or other suggestions?
If I understnd you correctly, you wished originally to perform Western blotting on proteins extracted from tissue silices, but you found that the amount of protein was too high to be loaded in a single well. Why can’t you just reduce the amont of extract loaded and perform Western blotting as usual ?
Hi, Why not just evaporate your samples to the correct volume? If they are not in native conditions, protein samples in the loading buffer are usually heated to 95C. At the same time the lid of the tube can be left open and reduce the samples to the correct volume.
If you really want to extract proteins from the gel, you should consider dialysis in the appropriate buffer with membranes having appropriate cut offs.
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