Analyzing protein biotinylation via Streptavidin gel shift

This assay utilizes the tight interaction between biotin and streptavidin to analyze what percentage of your protein of interest has been biotinylated.

Contents

1. Protocol

1. Protocol

WARNING: This is a general protocol, so you may need to change setup depending on the concentration of your protein and/or the dilutions of your streptavidin. Volumes and concentrations will change, but the general procedure will stay the same.

1. Pipette your protein of interest in a good concentration for coomassie stain into a set of tubes at the same volume, preferably a volume that works with your SDS loading buffer. For instance I have 5X, so I like to do 20uL(19uL protein and 1uL strep) and load 25.
2. Pick a buffer to dilute streptavidin in (I used 25mM Tris 8 with 100mM NaCL), but it doesn’t matter that much.
3. Add 1uL of either the buffer by itself or a dilution of streptavidin into each of the tubes, so that you get -, 1:1000, 1:100, 1:10, etc. as many as you’d like. It’s nice to have a tube that has just streptavidin for comparison.
4. Incubate the protein with streptavidin for about 15-20mins on ice or not, it doesn’t matter that much because streptavidin is pretty stable and you are denaturing your protein anyway.
5. Add SDS loading buffer. Do Not Boil as this will break the strep-bio bond.
6. Run the samples on an appropriate SDS PAGE gel so that you can see your protein and also your protein +75kDa because that is about what streptavidin runs at. You want to run the gel so that you can clearly resolve your protein, streptavidin (which will be very large because it is so concentrated), and your shifted protein.