Hi,
I currently have punched mouse brain tissue separated by brain region (using the cryostat) and the initial plan was to do Western Blot analysis on the punched tissue. We already have the protein antibodies ready, but it does not seem like Western blot is our best option to detect our proteins of interest (dopamine receptors). After BCA protein assay, we found that the calculated loading scheme amounts for each brain region exceeds the maximum well capacity of 15 ug. Does anyone have any troubleshoot mechanisms or other alternatives to analyze protein concentrations of punched samples (not Western Blot)? Preferably, a method that can detect multiple proteins of interest even with low protein concentrations. Or maybe a different method of protein extraction for punched samples? Thank you!
If you load the maximum reasonable volume in your wells, what is the highest amount of protein you can load equivalently across all wells? Western blot can be quite sensitive depending on the development method/reagents, the quality of the primary antibody and the expression level of your protein of interest. I've routinely detected bands from less than 1 ug total loaded protein.
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