Tetrahymena telomerase holoenzyme was purified as previously described (Jiang et al., 2013 (link)) with minor modifications. Fourteen liters of Tetrahymena TERT-FZZ cells were grown to ~400,000 cells/ml in PPYS. Cells were harvested by centrifugation, washed with 20 mM HEPES, pH 8.0, and lysed in detergent at 4 °C as described (Jiang et al., 2013 (link)). Lysate was clarified by ultracentrifugation and rabbit IgG-agarose resin (Sigma) was applied to supernatant. After binding, elution was performed by TEV protease. The TEV eluate was bound to anti-FLAG resin (Sigma) and eluted with 1.2 mL of 0.4 mg/ml FLAG peptide (Sigma) in buffer with 0.025% IGEPAL CA-630 (Sigma-Aldrich). To generate the DNA bound telomerase sample, the procedure described above was used with two modifications: 1µM of the DNA primer dGTTGGGGTTGGGGTLTLGLGLG (where superscript L is LNA) (Exiqon) and 10µM dGTP were added to the sample during the TEV elution step to saturate telomerase with DNA primer (Jiang et al., 2015 (link)). The final FLAG elution step was slightly modified for the cryoEM sample: a concentrated and detergent-free sample was generated by eluting from anti-FLAG resin using a small volume (25–50 µl) of 1mg/ml 3×FLAG peptide dissolved in elution buffer (20 mM HEPES•NaOH, pH 8.0, 50 mM NaCl, 1 mM MgCl2, 1 mM TCEP•HCl) supplemented with 50 µg/ml bacitracin.
Rabbit igg agarose resin
Manufactured by Merck Group
Rabbit IgG-agarose resin is a chromatography resin used for the purification of antibodies and other proteins. It consists of rabbit immunoglobulin G (IgG) covalently coupled to an agarose matrix. This resin is commonly used for the affinity-based purification of antibodies from complex biological samples.
Automatically generated - may contain errors
3 protocols using rabbit igg agarose resin
1
Purification of Tetrahymena Telomerase
2
Dad1 Protein Purification and Mass Spec
3
Purification of Plasma Membrane Proteins
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Plasma membrane
protein (100–500
μg) was solubilized with 10:1 (w/w) n-dodecyl-β-d -maltoside in resuspension buffer for 1 h at 4 °C. Unsolubilized
material was pelleted in a benchtop microcentrifuge at full speed
for 30 min at 4 °C. Solubilized upper-phase protein (0.1 mg,
100–200 μL total volume) was incubated for 1 h at 4 °C
with 15 μL of rabbit IgG agarose resin (Sigma, A9294) or 10
μL of IgG Sepharose 6 Fast Flow resin (GE Life Sciences, 17-0969-01)
equilibrated in resuspension buffer with 0.1% (w/v) DDM. Resin was
pelleted at 0.8 rcf for 1 min, and supernatant was discarded. Resin
was washed two times at 4 °C for 10 min with a 100× volume
of resuspension buffer containing 1% NP40 followed by a final wash
in a 100× volume of resuspension buffer containing 1% NP40 and
0.01% SDS. Resin was pelleted for 1 min at 0.8 rcf between each wash,
and supernatant was discarded. Protein was eluted overnight at 4 °C
with 30 units AcTEV protease (Invitrogen).
protein (100–500
μg) was solubilized with 10:1 (w/w) n-dodecyl-β-
material was pelleted in a benchtop microcentrifuge at full speed
for 30 min at 4 °C. Solubilized upper-phase protein (0.1 mg,
100–200 μL total volume) was incubated for 1 h at 4 °C
with 15 μL of rabbit IgG agarose resin (Sigma, A9294) or 10
μL of IgG Sepharose 6 Fast Flow resin (GE Life Sciences, 17-0969-01)
equilibrated in resuspension buffer with 0.1% (w/v) DDM. Resin was
pelleted at 0.8 rcf for 1 min, and supernatant was discarded. Resin
was washed two times at 4 °C for 10 min with a 100× volume
of resuspension buffer containing 1% NP40 followed by a final wash
in a 100× volume of resuspension buffer containing 1% NP40 and
0.01% SDS. Resin was pelleted for 1 min at 0.8 rcf between each wash,
and supernatant was discarded. Protein was eluted overnight at 4 °C
with 30 units AcTEV protease (Invitrogen).
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