Shreffler:Arah2 tetramers

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(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 13:33, 21 March 2012

Contents

Overview

This protocol is adapted from those published on the NIH tetramer core website with input from Jim Moon here at CIID

Materials

Equipment

  • test

Reagents

  • test

Procedure

Biotinylation Assay (to gauge whether SA is in excess) Procedure

  1. Add 5 µl of the Arah2-biotin monomer sample (0.5 mg/ml) to 12 µl of 1X PBS
  2. Label three Eppendorf tubes as "2X", “1X”, and "-" and add 5 µl of the Arah2-biotin monomer preparation.
  3. Add 5 µl of 0.8-1mg/ml Streptavidin to the tube labeled "2X".
  4. Add 2.5 µl of 0.8-1mg/ml Streptavidin and 2.5 µl of ddH2O to the tube labeled “1X”
  5. Add 5 µl of ddH2O to the tube labeled "-".
  6. Mix completely but gently.
  7. Incubate samples at room temperature for one hour.
  8. Add 10 µl of 2X loading buffer (non-reducing) to each sample.
  9. Do not boil or add DTT to either sample.
  10. Prepare and run samples on a 12% SDS-page gel.
  11. Stain the gel with 20% acetic acid and 0.1% Commassie blue in 100% methanol.
  12. Destain the gel using 40% EtOH and 10% acetic acid.


Discussion

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References

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