Question

You plan on isolating a protein of interest by cutting a band out of a gel. Your protein is a phosphatase (a certain type of enzyme) and you hope to measure its activity. Should you use SDS-PAGE or a native gel to isolate your protein? Why?

Answer 1

Everything will work if you are able to visualize the band in the gel before recovery, but the most important issue is what you want to do with the recovered protein. If you are interested in trying to recover functional protein then you are far better off not using an SDS gel in the first place. Bio-Rad makes an apparatus specifically for running native tube gels and these are actually easier to recover protein from than slabs. The process involves sectioning the tube gel with a purpose-built device that holds multiple razor blades that can be spaced in increments of 1 mm from each other, allowing up to about 100 slices from a 10 cm gel. However, in practice, slices are usually cut at 2-3 mm thickness to avoid crushing the gel during cutting and for ease of handling. The recovered slices are then individually crushed and eluted and activity assays can then be run on the extracts.

If, on the other hand, you have to use SDS-Page for some reason, and you are trying to recover functional protein, you will need to visualize and recover your band, and then remove the SDS and renature your protein (typically SDS is removed by acetone precipitation and the recovered protein is usually renatured in a 6M Guanidine-HCl solution - there are numerous protocols in the literature. If on the other hand, you are simply interested in recovering isolated protein for sequence analysis, you should blot it to PVDF, stain, and cut the band of interest from the blot with a razor or scalple.