X. laevis HSS was prepared as previously described (Lebofsky et al., 2009 (link)). Aliquots were snap frozen and thawed as necessary. To immunodeplete SSRP1, HSS diluted 1:10 in Unloading Buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.01% Tween) was exposed to two rounds of SSRP1 antibody. For each round, 150 μl of Dynabeads™ Protein G (Thermo Fisher Scientific) was washed three times in PBS, resuspended in 35 μg of SSRP1 antibody (Santa Cruz Biotechnology # sc-74536) and 825 μl of PBS, incubated with rotation for 90 min at room temperature, washed three times with PBST, resuspended in 2.4 μl of HSS diluted 1:10 in Unloading Buffer to 24 μl, and mixed for 45 min at room temperature. SPT16 antibody was generously provided by Dr. Hasan Yardimci. To immunodeplete SPT16, HSS was incubated with two rounds of 200 μl SPT16 antibody conjugated to 150 μl of Dynabeads™ Protein G. Mock depletions were conducted with the same amount of IgG antibody (BioLegend #400101).
Dynabeads protein g
Manufactured by BioLegend
Dynabeads™ Protein G are uniform, superparamagnetic beads coated with recombinant Protein G. Protein G is a bacterial surface protein that binds to the Fc region of immunoglobulins. These beads can be used for the isolation and purification of antibodies from complex biological samples.
Automatically generated - may contain errors
2 protocols using dynabeads protein g
1
Immunodepletion of SSRP1 and SPT16 from X. laevis
2
Immunoprecipitation and Western Blotting for Protein Interactions
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Cells were harvested 24 h after transfection and lysed for 30 min at 4°C in lysis buffer (50 mM Tris-HCl, pH 7.6, 2 mM EDTA, 150 mM NaCl, 0.5% NP-40) complemented with protease inhibitor cocktail. The cell lysates were immunoprecipitated with anti-FLAG M2 affinity Gel (Sigma-Aldrich) or anti-BST2 (2E6) antibody or the corresponding IgG isotype (Biolegend) coupled to Dynabeads® Protein G for 4 h at 4°C. Purified proteins were resolved by SDS-PAGE followed by western blotting using anti-HA conjugated to horseradish peroxidase (HRP) (Roche© Diagnostics), anti-FLAG®M2 conjugated to HRP (Sigma-Aldrich), anti-GFP conjugated to HRP (Genetex), anti-BST2 (National Institutes of Health) or anti-NEDD4 (Millipore) antibodies.
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