For immunoprecipitation 0.5 mg of total protein, 1 μg of homemade monoclonal EGFR antibody (clone 108.1; has been characterized before [44 (link)]) and 5 μg of monoclonal HER2 antibody (clone 13D1B1; has been characterized before [44 (link)]) together with 10 μl of protein A-sepharose (GE Healthcare) were used. For the HGF immunoprecipitation 1 ml of HL60 and MAD-NT cell CM and 20 μg monoclonal HGF antibody (R&D Systems) together with 15 μl of protein A-sepharose were used. For immunoblotting the following antibodies were used: p-tyrosine (homemade clone 4G10), pAkt S473 (Cell Signaling Technologies), pErk1/2 T202/Y204 (Cell Signaling Technologies), pSAPK/JNK T183/Y185 (Cell Signaling Technologies), Erk1/2 (Santa Cruz Biotechnology), EGFR (Cell Signaling Technologies), HER2 (Millipore), HGF (R&D Systems), pMet Y1234/Y1235 (Cell Signaling Technologies), pp38 T180/Y182 (Cell Signaling Technologies) and tubulin (Sigma).
Protein a sepharose
Manufactured by R&D Systems
Sourced in United States
Protein A-Sepharose is a chromatography resin used for the purification of antibodies and immunoglobulins. It consists of Protein A, a bacterial protein that binds to the Fc region of antibodies, coupled to cross-linked agarose beads. The resin can be used in column chromatography to selectively capture and purify antibodies from complex mixtures, such as cell culture supernatants or serum samples.
Automatically generated - may contain errors
Lab products found in correlation
Tubulin, by Merck Group (1 mentions)
P sapk jnk t183 y185, by Cell Signaling Technology (1 mentions)
P erk1 2 t202 y204, by Cell Signaling Technology (1 mentions)
P p38 t180 y182, by Cell Signaling Technology (1 mentions)
P tyrosine, by Cell Signaling Technology (1 mentions)
P met y1234 y1235, by Cell Signaling Technology (1 mentions)
Erk1 2, by Santa Cruz Biotechnology (1 mentions)
Pakt s473, by Cell Signaling Technology (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
Hepatocyte growth factor (hgf), by R&D Systems (1 mentions)
2 protocols using protein a sepharose
1
Receptor Tyrosine Kinase Signaling Assay
2
Co-immunoprecipitation of 5-HT2A and TrkB
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Co-immunoprecipitation in lysates of N1E-115 cells that co-expressed HA-tagged 5-HT2A and GFP-tagged TrkB was performed as described previously [35 (link)] with an antibody against GFP (1:250; GeneTex, cat. # GTX26556, RRID:AB_371421). Immunoblotting was carried out using a horseradish peroxidase (HRP)-conjugated anti-GFP (1:1000; LSBio (LifeSpan, Providence, RI, USA), cat. # LS-C50850-500, RRID:AB_1220053) or an HRP-conjugated anti-HA tag (1:250; Roche, Basel, Switzerland, cat. # 12013819001, RRID:AB_390917) antibody.
Co-immunoprecipitation from hippocampal, cortical, and striatal homogenates was performed according to a protocol described elsewhere [39 (link)]. Briefly, brain samples isolated from adult (P90) C57BL/6J mice were homogenized, and membrane fractions were prepared by differential centrifugation. The lysates were incubated with a goat polyclonal antibody against 5-HT2A (1:50; Santa Cruz Biotechnology, Dallas, TX, USA, cat. # sc-15073, RRID:AB_2119724) followed by incubation with Protein A-Sepharose and Western blotting with an anti-TrkB antibody (1:1000; R&D Systems, Minneapolis, MN, USA, cat. # AF1494, RRID:AB_2155264).
Co-immunoprecipitation from hippocampal, cortical, and striatal homogenates was performed according to a protocol described elsewhere [39 (link)]. Briefly, brain samples isolated from adult (P90) C57BL/6J mice were homogenized, and membrane fractions were prepared by differential centrifugation. The lysates were incubated with a goat polyclonal antibody against 5-HT2A (1:50; Santa Cruz Biotechnology, Dallas, TX, USA, cat. # sc-15073, RRID:AB_2119724) followed by incubation with Protein A-Sepharose and Western blotting with an anti-TrkB antibody (1:1000; R&D Systems, Minneapolis, MN, USA, cat. # AF1494, RRID:AB_2155264).
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