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Wayne G. Shreffler (Talk | contribs)
(New page: ==Overview== This protocol is adapted from those published on the NIH tetramer core [http://tetramer.yerkes.emory.edu/client/protocols#10 website] with input from [http://www.massgeneral.o...)
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Revision as of 14:33, 21 March 2012
Biotinylation Assay (to gauge whether SA is in excess) Procedure
- Add 5 µl of the Arah2-biotin monomer sample (0.5 mg/ml) to 12 µl of 1X PBS
- Label three Eppendorf tubes as "2X", “1X”, and "-" and add 5 µl of the Arah2-biotin monomer preparation.
- Add 5 µl of 0.8-1mg/ml Streptavidin to the tube labeled "2X".
- Add 2.5 µl of 0.8-1mg/ml Streptavidin and 2.5 µl of ddH2O to the tube labeled “1X”
- Add 5 µl of ddH2O to the tube labeled "-".
- Mix completely but gently.
- Incubate samples at room temperature for one hour.
- Add 10 µl of 2X loading buffer (non-reducing) to each sample.
- Do not boil or add DTT to either sample.
- Prepare and run samples on a 12% SDS-page gel.
- Stain the gel with 20% acetic acid and 0.1% Commassie blue in 100% methanol.
- Destain the gel using 40% EtOH and 10% acetic acid.
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare