<p>Anti-GFP Binding Magnetic Beads</p><p>Antibody Type: Recombinant antibody Isotype: N/A Immunogen: Green Fluorescent Protein Lot: 130628 Lot Binding Capacity: 1ug/uL bead slurry Formulation: 50% slurry of beads in PBS + 0.5% Sodium Azide</p><p>APPLICATIONS</p><p>Assay ELISA WB IP IF IHC Recommended No No 5uL slurry No No Concentration per 1ug target protein Note: Optimal dilutions should be established by the end user. These concentrations are intended as a guide.</p><p>LIGAND COUPLING STABILITY AND TARGET ELUTION EFFICIENCY</p><p>Elution Conditions Magnetic Beads Sepharose Beads Bound Ligand Target Elution Bound Ligand Target Elution Leakage Efficiency (i.e. GFP) Leakage Efficiency (i.e. GFP) Reducing buffer 1-3% 100% <0.05% 100% + Boiling Non-reducing buffer+ <1% ~75-95% < 0.005% ~75-95% Boiling Low pH: <2% ~25-35% BDL ~25-35% Glycine pH 2.0 High salt: BDL ~15-20% BDL ~15-20% 3.6 M MgCl</p><p>Bound ligand leakage = the estimated percentage of GBP protein that will be present in the elute using the indicated elution conditions. Target Elution Efficiency = the estimated percentage of bound GFP target protein that was eluted using the indicated elution conditions. GFP eluted using Reducing buffer + boiling was considered 100%, all others were quantified relative to this condition. BDL= Below Detection Limits (0.001%)</p><p> www.vanderbilt.edu/vapr Phone: 936-3092 Room: 892 PRB 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. </p><p>Protocol 1. Thoroughly mix beads and storage buffer 2. Remove desired amount of resin bead slurry 3. Place beads on magnetic stand and remove storage buffer 4. Wash beads 3X with 500uLs of buffer being used for IP (i.e. RIPA lysis buffer) 5. Each time: vortexing, placing on magnetic stand, and removing buffer 6. After second wash, resuspend beads in IP buffer to original bead volume 7. Add in GFP containing lysate/buffer/etc. 8. Rotate end-over-end for 1 hour at room temperature or overnight at 4°C 9. Place bead tube on magnetic rack 10.Remove unbound protein in solution for use as flow-through sample 11.Wash beads in lysis buffer 3X 12.If necessary retain washes to examine on SDS-PAGE. 13.Elution conditions will vary across experiments. Four different elution conditions have been tested in VAPR and their data is shown above on page 1. 14.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. 15.If eluting via Glycine or High Salt, a. add 3 volumes of elution buffer and incubate for 3 to 5 minutes. b. Place on magnetic stand c. 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.</p><p>REFERENCES</p><p>STORAGE AND UTILIZATION</p><p>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. </p><p>All VAPR products are quality tested and fully guaranteed. If you have any issues please contact us.</p><p> www.vanderbilt.edu/vapr Phone: 936-3092 Room: 892 PRB</p>