p300-BRPHZ and p300-ZZ cloned in the pGEX-6P-1 vector were expressed in BL21 Rosetta 2 cells. Protein production was induced with 0.2 mM IPTG and cultured overnight at 16 °C in Luria broth (LB) medium supplemented with 0.05 mM ZnCl2. The GST-tagged proteins were purified on glutathione Sepharose 4B beads (Amersham) in binding buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40, 1 mM PMSF plus protease inhibitors (Roche)) and eluted by 100 mM Tris pH 8.0 containing 15 mg/mL Reduced Glutathione (Sigma). All proteins harboring mutations or deletions were expressed and purified as WT proteins.
Sepharose 4b beads
Manufactured by Cytiva
Sepharose 4B beads are a type of agarose-based chromatography resin used for protein purification and separation. They provide a porous, hydrophilic matrix with a high chemical and physical stability. Sepharose 4B beads are commonly used in size exclusion, ion exchange, and affinity chromatography techniques.
Automatically generated - may contain errors
Lab products found in correlation
Reduced glutathione, by Merck Group (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Anti sqstm1 p62, by Abnova (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti myc antibody, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Anti lc3, by Nanotools (1 mentions)
Ant flag, by Merck Group (1 mentions)
Anti myc, by Santa Cruz Biotechnology (1 mentions)
4 protocols using sepharose 4b beads
1
Purification of GST-tagged p300 Proteins
2
Immunoprecipitation and Western Blot Analysis of Autophagy Proteins
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The following antibodies were used for immunoprecipitation and western blot analysis: anti-human FYCO1 (Abnova, H00079443-A01), anti-LC3 (Nanotools, 5F10), anti-p62/SQSTM1 (Abnova, 2C11), anti-αA-crystallin (Santa Cruz Biotechnology, sc-22743), anti-αB-crystallin (Acris, AP20218PU-N), ant-FLAG (Sigma, M2), anti-Myc (Santa Cruz Biotechnology, 9E10), anti-GFP (MBL, 598), anti-actin (Santa Cruz Biotechnology, sc-1615), anti-Myc antibody (Santa Cruz Biotechnology, 9E10), anti-FLAG antibody (Sigma, M2) and anti-GAPDH (Santa Cruz Biotechnology, sc-32233). An antibody against mouse FYCO1 was generated by immunizing rabbits with a peptide containing amino acids 1258–1274 of mouse FYCO1, and was affinity-purified using the immunizing antigen immobilized on CNBr-activated Sepharose 4B beads (Amersham).
3
Purification of GST-tagged p300 Proteins
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p300-BRPHZ and p300-ZZ cloned in the pGEX-6P-1 vector were expressed in BL21 Rosetta 2 cells. Protein production was induced with 0.2 mM IPTG and cultured overnight at 16 °C in Luria broth (LB) medium supplemented with 0.05 mM ZnCl2. The GST-tagged proteins were purified on glutathione Sepharose 4B beads (Amersham) in binding buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40, 1 mM PMSF plus protease inhibitors (Roche)) and eluted by 100 mM Tris pH 8.0 containing 15 mg/mL Reduced Glutathione (Sigma). All proteins harboring mutations or deletions were expressed and purified as WT proteins.
4
Recombinant Protein Covalent Binding
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To confirm covalent binding of selected TPs identified in cell extracts with recombinant proteins, PfLDH86 (link) and PfSAHH47 were chosen as representative interaction partners of PfPrx1a wt. For these experiments, 2 mg of reduced recombinant PfPrx1a wt was coupled to CNBr-activated Sepharose 4B beads (Amersham) as described above in the pull-down implementation chapter and was kept in a reduced state with 0.5 mM DTT during all immobilization steps (see above). After a final washing of the beads in the absence of DTT, 8 mg purified recombinant PfLDH wt or PfSAHH wt (not pre-reduced) were incubated with the loaded beads. As described above for the pull-downs with cell lysates (please see section above for details), the beads were extensively washed, and bound protein was eluted with 10 mM of DTT and visualized via silver staining on SDS gels.
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