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...)
(Procedure)
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==Procedure==
==Procedure==
-
Biotinylation Assay (to gauge whether SA is in excess)
+
===Biotinylation Assay===
 +
 
 +
Background
 +
To gauge whether SA is in excess. Can be used to modify step below. Non-reducing gel required to determine uncoordinated Arah2 and SA by MW
 +
 
Procedure
Procedure
# Add 5 µl of the Arah2-biotin monomer sample (0.5 mg/ml) to 12 µl of 1X PBS
# Add 5 µl of the Arah2-biotin monomer sample (0.5 mg/ml) to 12 µl of 1X PBS
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# Destain the gel using 40% EtOH and 10% acetic acid.
# Destain the gel using 40% EtOH and 10% acetic acid.
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===Tetramerization===
 +
Background
 +
 +
The following information is a generalization of how much labeled streptavidin to add to the biotinylated monomers at the last stage of the tetramer preparation. The actual amount of streptavidin to be added depends both on the molecular weight of the monomer as well as the percent biotinylation. In the Altman Lab, this amount will be calculated for you in the "JDA Tetramer stocks" database and given as a total volume to be added to the monomer solution. As such, it is necessary to divide this amount by ten to arrive at the amount to be added per time interval.
 +
 +
The procedure of adding the streptavidin over 10 time intervals is designed to maximize tetramer formation. At early additions, the biotinylated MHC will be in excess and will saturate all of the avidin. At the conclusion of the streptavidin addition, there may be an excess of avidin, however, the assumption is that most of the MHC will have gone into tetramers. If the streptavidin was added at one time and was in excess, the resulting solution would be more likely to exist as a mixture of monomers/dimers/trimers.
 +
 +
Comments on stretpavidin addition protocol
 +
*After step 1, biotinylated monomer is present in vast excess, and all the biotin binding sites on the added streptavidin are occupied with MHC monomer
 +
*After step 2, biotinylated monomer is still present in excess, and all the biotin binding sites on the added streptavidin are occupied with MHC monomer.
 +
*After step 9, the concentrations of biotinylated monomer and biotin binding sites on the streptavidin are nearly identical. Note that for this example, we have consciously overestimated the concentration of biotinylated monomer. Normally, we try to shoot for "equimolarity" at step 10.
 +
*The addition of streptavidin in steps 10 adds excess streptavidin, which remains free. The excess streptavidin will not bind to cells when used in staining reactions. Of course, it could increase the staining background, but it won't be in vast excess, so its contribution to the background noise will be minimal.
==Discussion==
==Discussion==

Revision as of 13:37, 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

Background To gauge whether SA is in excess. Can be used to modify step below. Non-reducing gel required to determine uncoordinated Arah2 and SA by MW

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.

Tetramerization

Background

The following information is a generalization of how much labeled streptavidin to add to the biotinylated monomers at the last stage of the tetramer preparation. The actual amount of streptavidin to be added depends both on the molecular weight of the monomer as well as the percent biotinylation. In the Altman Lab, this amount will be calculated for you in the "JDA Tetramer stocks" database and given as a total volume to be added to the monomer solution. As such, it is necessary to divide this amount by ten to arrive at the amount to be added per time interval.

The procedure of adding the streptavidin over 10 time intervals is designed to maximize tetramer formation. At early additions, the biotinylated MHC will be in excess and will saturate all of the avidin. At the conclusion of the streptavidin addition, there may be an excess of avidin, however, the assumption is that most of the MHC will have gone into tetramers. If the streptavidin was added at one time and was in excess, the resulting solution would be more likely to exist as a mixture of monomers/dimers/trimers.

Comments on stretpavidin addition protocol

  • After step 1, biotinylated monomer is present in vast excess, and all the biotin binding sites on the added streptavidin are occupied with MHC monomer
  • After step 2, biotinylated monomer is still present in excess, and all the biotin binding sites on the added streptavidin are occupied with MHC monomer.
  • After step 9, the concentrations of biotinylated monomer and biotin binding sites on the streptavidin are nearly identical. Note that for this example, we have consciously overestimated the concentration of biotinylated monomer. Normally, we try to shoot for "equimolarity" at step 10.
  • The addition of streptavidin in steps 10 adds excess streptavidin, which remains free. The excess streptavidin will not bind to cells when used in staining reactions. Of course, it could increase the staining background, but it won't be in vast excess, so its contribution to the background noise will be minimal.

Discussion

discuss this protocol

References

Contact

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