Phone: 936-3092 Room: 892 PRB

www.vanderbilt.edu/vapr Phone: 936-3092 Room: 892 PRB

GST Binding Sepharose Resin

www.vanderbilt.edu/vapr Phone: 936-3092 Room: 892 PRB

Antibody Type: Recombinant antibody

Isotype: N/A

Immunogen: GST

Lot: 130628

Lot Binding Capacity: 0.5mg/ml settled resin

Formulation: 50% slurry of resin in PBS + 0.5% Sodium Azide

www.vanderbilt.edu/vapr Phone: 936-3092 Room: 892 PRB

APPLICATIONS

Assay / ELISA / WB / IP / IF / IHC
Recommended
Concentration / No / No / 1ml settled resin per 0.5mg target protein / No / No

Note: Optimal dilutions should be established by the end user. These concentrations are intended as a guide.

www.vanderbilt.edu/vapr Phone: 936-3092 Room: 892 PRB

Ligand Coupling stability and Target Elution Efficiency

Elution Conditions / Magnetic Beads / Sepharose Beads
Bound Ligand Leakage / Target Elution Efficiency (i.e. GST) / Bound Ligand Leakage / Target Elution Efficiency (i.e. GST)
Reducing buffer
+ Boiling / 1-3% / 100% / <0.05% / 100%
Non-reducing buffer+ Boiling / <1% / ~75-95% / < 0.005% / ~75-95%
Low pH:
Glycine pH 2.0 / <2% / ~25-35% / BDL / ~25-35%
High salt:
3.6 M MgCl / BDL / ~15-20% / BDL / ~15-20%

Bound ligand leakage = the estimated percentage of GST protein that will be present in the elute using the indicated elution conditions.

Target Elution Efficiency = the estimated percentage of bound GST target protein that was eluted using the indicated elution conditions. GST eluted using Reducing buffer + boiling was considered 100%, all others were quantified relative to this condition.

BDL= Below Detection Limits (0.001%)

SUGGESTED PROTOCOL

Note: The following is a standard Immunoprecipitation protocol and may need to be adjusted based on the user’s experimental conditions. The theoretical binding capacity is 4mg/ml of settled resin. However, in practice we have generally found the binding capacity to be approximately 0.5mg/ml of settled resin.

Protocol

  1. Thoroughly mix beads and storage buffer
  2. Remove desired amount of resin (mixed resin:buffer is a 50:50 slurry)
  3. Centrifuge resin slurry at 1,000 x g for 2 minutes
  4. Remove storage buffer
  5. Wash with 5 volumes of PBS where the volume represents amount of settled resin
  6. Centrifuge resin slurry at 1,000 x g for 2 minutes
  7. Remove PBS
  8. Add in GST containing lysate/buffer/etc.
  9. Rotate end-over-end for 1 hour at room temperature or 2 hours at 4°C
  10. Centrifuge sample at 1,000 x g for 2 minutes
  11. Remove unbound protein in solution for use as flow-through sample
  12. Wash beads with 5 volumes of PBS (or appropriate wash buffer)
  13. Centrifuge resin slurry at 1,000 x g for 2 minutes
  14. Remove wash buffer
  15. Repeat steps 12 through 14 two times. If necessary retain washes to examine on SDS-PAGE.
  16. Elution conditions will vary across experiments. Four different elution conditions have been tested in VAPR and their data is shown above on page 1.
  17. If using Protein Gel Loading Buffer as the elution strategy add desired volume of Sample Loading Buffer and proceed with boiling and/or gel loading.
  18. If eluting via Glycine or High Salt,
  19. add 3 volumes of elution buffer and incubate for 3 to 5 minutes.
  20. Centrifuge at 1,000 x g for 2 minutes
  21. Remove eluted sample. Please note that Glycine eluted samples with need to have their pH neutralized and High Salt eluted samples will need to be buffer exchanged before running on SDS-PAGE.

REFERENCES

STORAGE AND UTILIZATION

Antibodies bound to solid matrices are provided in PBS with 0.05% Sodium azide. Stored at 4˚C, the solution will be stable for 6 months or longer.

All VAPR products are quality tested and fully guaranteed.

If you have any issues please contact us.

www.vanderbilt.edu/vapr Phone: 936-3092 Room: 892 PRB

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