I am trying to isolate livers from 5 day old zebrafish for western, but can't get enough protein. I already maximize the no. of fish I can humanly dissect. Is there a way to do western on very little amount of protein?
- If you try ECL detection and use ECL prime detection (GE healthcare) you can use
What about flow cytometry?
Using antibody staining, you can detect expression of your protein of interest on a cell by cell basis. And you will obtain a histogram instead of a single band.
Also, you do not have to worry about loading equal amounts of protein into each well, since you will be testing protein expression on a single-cell basis. Finally, you can quantify a number of your proteins per cell. We routinely use Quantum™ Simply Cellular® microspheres from http://www.bangslabs.com/products/flow-cytometry/fluorescence-quantitation. For example see Fig. 3 in https://www.ncbi.nlm.nih.gov/pubmed/19251697. Good luck.
Dear Alex,
Flow cytometry won't work. I want to check autophagy by detecting the conversion of LC3B I to LC3B II. The quantity of the LC3B protein is not important.
Thank you.
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