Tissues were homogenized as described in the Western Analysis section, and 100 μg extract was diluted into 1 ml of RIPA buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) containing protease and phosphatase inhibitor cocktails (Thermo Scientific, Waltham, MA). Diluted extracts were precleared for 3 hr by incubation with 20 μl protein A-agarose (Thermo Scientific, Waltham, MA), followed by overnight incubation (on a rotator) in 20 μl protein A-agarose and 5 μg phospho-TH (ser40) antibody at 4°C. Agarose beads were collected by centrifugation (5 min, 1,000 x g) and washed five times with RIPA buffer. Following the final wash and bead collection, the bound proteins were removed by incubation in 1X Wes sample buffer for 10 min at 65°C. These extracts were then used for western analysis to examine K63-linked ubiquitination and TH levels.
Protein a agarose
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Protein A agarose is a chromatography resin composed of protein A, a bacterial protein, immobilized on agarose beads. It is commonly used for the purification and isolation of antibodies from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Expi293f cells, by Thermo Fisher Scientific (6 mentions)
Protein a agarose, by GE Healthcare (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Expi293 cell, by Thermo Fisher Scientific (3 mentions)
Protein g agarose, by Thermo Fisher Scientific (3 mentions)
Igg elution buffer, by Thermo Fisher Scientific (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Protein a agarose, by Santa Cruz Biotechnology (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (3 mentions)
Superdex 75 column, by GE Healthcare (2 mentions)
Superdex 200 increase 10 300 column, by GE Healthcare (2 mentions)
Captureselect kappa xl affinity matrix, by Thermo Fisher Scientific (2 mentions)
35s methionine cysteine, by PerkinElmer (2 mentions)
Endoglycosidase h, by New England Biolabs (2 mentions)
Pngase f, by New England Biolabs (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Amicon ultra 4 100k, by Merck Group (2 mentions)
Igg binding buffer, by Thermo Fisher Scientific (2 mentions)
127 protocols using protein a agarose
1
Immunoprecipitation and Western Analysis of TH
2
Protein Purification Protocol
3
Protein-A Agarose Purification
4
Antibody Expression and Purification
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Plasmids encoding antibody heavy and light chains were transfected into Expi293 cells (Invitrogen). IgGs and Fabs were purified using Protein A agarose (Fisher) or CaptureSelect IgG-CH1 affinity matrix (Life Technologies), respectively.
5
Fab-Fc Antibody Purification
6
Fab-Fc Antibody Production and Purification
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The Fab region of the G2 heavy chain was fused with the HRV3C cleavage site followed by human IgG1 Fc fragment and subcloned into the eukaryotic expression vector pVRC8400. This plasmid (plasmid 1) was cotransfected with a vector encoding the G2 light chain (plasmid 2) into Expi293 cells (Invitrogen), and the secreted antibody was purified using Protein A agarose (Fisher). HRV3C protease (1% wt/wt) was added to the protein and the reaction was incubated for 2 h at room temperature. The digested antibody was passed back over Protein A agarose to remove the Fc fragment, and the unbound Fab was additionally purified using a Superdex 75 column (GE Healthcare).
7
Purification of Dsg Extracellular Domains
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Dsg1- and Dsg3-Fc constructs containing the full extracellular domain of the respective Dsg were stably expressed in Chinese hamster ovary cells (CHO-cells). Purification was performed as described elsewhere in detail (10 (link)). Briefly, transfected CHO-cells were grown to confluence, supernatants were collected and recombinant proteins were isolated using Protein A Agarose (Life Technologies). To test purity and specificity Coomassie staining and Western blotting using anti Dsg1-monoclonal antibody (mAb) (p124, Progen, Heidelberg, Germany) and anti Dsg3-mAb (clone5G11; Life Technologies) which both detect the extracellular domain of the respective Dsg were conducted (data not shown).
8
Production and Purification of PD-L1 Fusion Proteins
9
Recombinant Dsg1 and Dsg3 Purification
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Dsg1- and Dsg3-Fc constructs containing the full extracellular domain of the respective Dsg were stably expressed in Chinese Hamster Ovary cells (CHO cells) and purified using protein A agarose (Life Technologies). Purification was performed as described elsewhere in detail [24 (link), 25 ].
10
Chromatin Immunoprecipitation in Yeast
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Log-phase yeast cultures were fixed in 3% paraformaldehyde (Sigma-Aldrich) for 30 min on ice. The reaction was quenched with the addition of 0.0625 (v/v) of 2.5 M glycine (Thermo Fisher Scientific, Wilmington, DE, USA) and the samples washed twice in PBS, sonicated, and lysed. The samples were then immunoprecipitated with specific antibodies, which were later bound to protein A agarose (Life Technologies, Carlsbad, CA) and protein G agarose (Roche Diagnostic, Indianapolis, IN) (1:1). The agarose was washed and reverse crosslinked by incubating for >8 h at 65°C. The immunoprecipitants were then digested with 20 mg/ml Proteinase K (Roche Diagnostics, Basel, Switzerland) for 2 h at 37°C and extracted with 1 vol phenol:chloroform:isoamyl alcohol (25:24:1) (Nacalai Tesque, Kyoto, Japan). The aqueous layer was then ethanol precipitated, dried, and analyzed with PCR using centromere-specific primers (see Supplementary Table S2 ). The previously published sonication method and buffer composition were closely followed (30 (link),41 (link)).
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