Transfected cells from the indicated number of 10-cm plates were lysed in 1ml per 10-cm plate of immunoprecipitation buffer (58 (link)) containing protease and phosphatase inhibitors (50mM HEPES pH 7.4, 100mM NaCl, 50mM NaF, 10mM NaH2PO4, 2mM EDTA, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 0.5μM microcystin, 1X Roche cOmplete EDTA-free Protease Inhibitors), aliquots of pre-cleared lysates were taken for loading controls, and the indicated proteins were immunoprecipitated with 2μl of rabbit 6325 antiserum (10 (link)) per 10-cm plate for GC-A proteins or 2μl of rabbit 6327 antiserum (11 (link)) per 10-cm plate for GC-B proteins and 25μl of a 50% slurry of Protein-A-agarose beads (Repligen) per 10-cm plate overnight. The beads were washed three times with immunoprecipitation buffer lacking NaCl. After boiling for 5 minutes in 2X reducing sample buffer, samples were fractionated on 8% resolving gels. The gel was first stained with ProQ-Diamond (Molecular Probes) and then stained with SYPRO Ruby protein stain (Molecular Probes) as previously described (38 (link)). Lysates used for full length GC-A or GC-B western blots were fractionated on 8% resolving gels, and lysates used for β-actin or soluble GC-A intracellular domains were fractionated on 10% resolving gels.
Protein a agarose beads
Manufactured by Repligen
Sourced in United States
Protein A agarose beads are a type of chromatography resin commonly used for the purification of monoclonal antibodies and other proteins. These beads are composed of agarose, a polysaccharide derived from seaweed, and immobilized Protein A, a bacterial cell wall protein that has a high affinity for the Fc region of immunoglobulins. The Protein A agarose beads provide a versatile and efficient platform for the capture and purification of target proteins from complex mixtures, such as cell culture supernatants or biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pro q diamond, by Thermo Fisher Scientific (1 mentions)
Sypro ruby protein stain, by Thermo Fisher Scientific (1 mentions)
25 kda linear polyethyleneimine, by Polysciences (1 mentions)
Hek293f cell, by Thermo Fisher Scientific (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Fish skin gelatin, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Sensimix sybr mix, by Meridian Bioscience (1 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Anti m6a antibody, by Synaptic Systems (1 mentions)
Rnasin, by Promega (1 mentions)
Glutathione sepharose 4b, by Cytiva (1 mentions)
Anti flag antibody, by Thermo Fisher Scientific (1 mentions)
Pierce protein g magnetic beads, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
35s methionine, by Hartmann Analytic (1 mentions)
16 protocols using protein a agarose beads
1
Immunoprecipitation and Phosphorylation Analysis of Guanylate Cyclase Proteins
2
Construction of Bispecific Antibody ERC6
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For construction of the bispecific cetuximab × anti-cotinine scFv antibody (ERC6) and negative control IgG × anti-cotinine scFv antibody (NCC6) expression vectors, genes encoding the light chain and heavy chain fused with an anti-cotinine single chain variable fragment (scFv) through a linker (Gly-Gly-Gly-Gly-Ser)4 were chemically synthesized (Genscript, Piscataway, NJ, USA). The restriction sites AgeΙ and XbaΙ were inserted at the 5′ and 3′ ends, respectively, of the gene encoding the light chain of cetuximab and the negative control antibody. Additional restriction sites, NheΙ and BsiWΙ, were inserted at the 5′ end of the genes encoding the heavy chain of cetuximab and negative control antibody and at the 3′ end of the genes encoding anti-cotinine scFv, respectively. Light chain and heavy chain/linker/anti-cotinine/scFv were subcloned into the mammalian expression vector designed for secretion of recombinant proteins, as described previously21 (link).
The expression vectors encoding ERC6 and NCC6 were transfected into HEK293F cells (Invitrogen) using 25-kDa linear polyethyleneimine (Polyscience, Warrington, PA, USA), as reported previously23 (link). ERC6 and NCC6 were purified from the culture supernatants by affinity chromatography using protein A agarose beads (RepliGen, Waltham, MA, USA) as described previously24 (link).
The expression vectors encoding ERC6 and NCC6 were transfected into HEK293F cells (Invitrogen) using 25-kDa linear polyethyleneimine (Polyscience, Warrington, PA, USA), as reported previously23 (link). ERC6 and NCC6 were purified from the culture supernatants by affinity chromatography using protein A agarose beads (RepliGen, Waltham, MA, USA) as described previously24 (link).
3
Immunoprecipitation and GTPase Pulldown Assays
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Cells were lysed in chilled immunoprecipitation buffer and the cleared lysate (500 μg) incubated with 3 μg of the indicated antibody and 30 μL of protein-A agarose beads (Repligen) in 1 mL of buffer. Beads were resuspended in 30 μL of 2X Laemmli buffer and subjected to Western blotting.
For GTP-pulldown assays, cell lysates were incubated with beads glutathione-S-transferase (GST)-Rhotekin Rho binding domain (RBD), GST-NDR1 or GST-NDR2 (Carna Biosciences, Japan), then precipitates analyzed by Western blotting using anti-RhoB, anti-GEF-H1 or anti-S885phospho-GEF-H1 antibodies.
For GTP-pulldown assays, cell lysates were incubated with beads glutathione-S-transferase (GST)-Rhotekin Rho binding domain (RBD), GST-NDR1 or GST-NDR2 (Carna Biosciences, Japan), then precipitates analyzed by Western blotting using anti-RhoB, anti-GEF-H1 or anti-S885phospho-GEF-H1 antibodies.
4
Immunoprecipitation of AZGP1 Protein
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Cells were lysed in 1ml of chilled RIPA buffer, and the cleared lysate (400 µl) was incubated with 5 µl of the indicated antibody (AZGP1, Sigma) and 20 µl of protein-A agarose beads (Repligen, USA). The reaction was brought up to a final volume of 500µl with IP buffer. After constant rotation for 3 hours, beads were resuspended in 2× SDS buffer and subjected to Western blot analysis.
5
Immunopurification of Ofd1 and Nro1
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Ofd1 and Nro1 immunopurifications (IPs) were performed as previously described (Lee et al., 2009 (link)) with modifications. Briefly, 1 × 108 cells (Nro1 IP) or 2 × 108 cells (Ofd1 IP) were pelleted, washed in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4 plus protease inhibitors) and resuspended in 1 ml PBS plus 2 mM DSP (Pierce, Waltham, MA) or vehicle (8% DMSO unless stated otherwise) for 5 min to crosslink proteins. The reaction was quenched by addition of 1 M Tris-HCl pH 7.5 to a final concentration of 20 mM Tris. Ribosome-cleared lysates were generated as described above and 1.0–1.5 mg protein in 600 μl volume was incubated with affinity-purified antibodies (8 ng antibody/μg protein) and 30 μl Protein A agarose beads (Repligen, Waltham, MA) for 2 hr at 4°C. Beads were washed three times and resuspended in SDS lysis buffer (10 mM Tris-HCl pH 6.8, 100 mM NaCl, 1% [w/v] SDS, 1 mM EDTA, 1 mM EGTA) plus protease inhibitors prior to boiling (95°C, 5 min) and immunoblotting.
6
Purification of CKM-Mediator Complex
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For purification of the CKM–Mediator complex from wild type or tamoxifen-treated Med13/13lfl/fl ESCs, 600 µg of nuclear extract was diluted in BC150 buffer (50 mM Hepes pH 7.9, 150 mM KCl, 0.5 mM EDTA, 0.5 mM DTT, cOmplete protease inhibitor cocktail (Roche)). Samples were incubated with 5 µg CDK8 antibody (A302-500A, Bethyl laboratories) and 25 units benzonase nuclease (Millipore) overnight at 4 °C. For purification of T7-MED14, 5 μl T7-Tag antibody (D9E1X, 13246, Cell Signaling) and 25 units benzonase nuclease were used. Protein A agarose beads (RepliGen) were blocked for 1 h at 4 °C in Buffer BC150 containing 1% fish skin gelatin (Sigma) and 0.2 mg ml-1 BSA (New England Biolabs). The blocked beads were added to the samples and incubated for 4 h at 4 °C. Four washes for 10 min each were performed using BC150 containing 0.02% NP-40. The beads were resuspended in 2× SDS loading buffer and boiled for 5 min to elute the immunoprecipitated complexes.
7
Co-Immunoprecipitation of RING1B Complex
8
Immunoprecipitation of HCMV Proteins
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MRC5 fibroblasts were infected and pulsed at 72 hpi with 35S-methionine for 6 h at 37 °C. Cells were lysed in NP40 and immunoprecipitated using neutralizing gB mAbs 2F4, 5A6, 7H7, 8H2 or gH mAbs 10C10 and 5C3. Anti-GFP antibody was used as an isotype control. Following incubation of immune complexes with protein A agarose beads (RepliGen), samples were treated with 1 × SDS sample buffer and resolved using SDS–PAGE. For experiments involving N-glycanase F treatment, following immunoprecipitation the protein A beads were split into 2 fractions and one fraction was incubated with 500 units of PNGase F (New England Biolabs) for 2 h before addition of sample buffer and resolution by SDS–PAGE.
9
PCGF2 and RYBP ChIP-qPCR
10
Immunoprecipitation of Phosphotyrosine Proteins
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