Cells from an exponentially growing S. solfataricus culture (OD600 of ~0.4) were harvested and resuspended in lysis buffer (50 mM Tris-HCl, pH 6.8, 150 mM NaCl, 0.1 mM DTT and 10% (v/v) glycerol). The cells were lysed either with 0.5% Triton X-100 or by sonication, and centrifuged at 30,000g for 20 min at 4°C. The supernatant was incubated for 30 min at 4°C with DNase I (100 U) and RNase A (100 U). Immunoprecipitation with indicated antibodies and subsequently with protein A beads was performed according to the manufacturer’s instruction (Clontech). The antibodies were prepared in rabbit with purified recombinant proteins as the antigens. The immunoprecipitates were resolved by SDS–PAGE, and the gel was stained with Coomassie brilliant blue G250. Proteins of interest were recovered from the gel and identified by liquid chromatography-tandem mass spectrometry. To detect protein–protein interactions, two tested proteins (10 μg each) were co-immunoprecipitated as described above. Rabbit antibodies against PriX, PriS or PriL were employed in the assays.
Protein a beads
Manufactured by Takara Bio
Protein A beads are a type of affinity chromatography resin used for the purification of antibodies. They consist of a cross-linked agarose matrix with covalently attached Protein A, a bacterial protein that binds to the Fc region of immunoglobulins. Protein A beads are commonly used for the capture and isolation of monoclonal and polyclonal antibodies from complex samples, such as cell culture supernatants or serum.
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Lab products found in correlation
Protein g agarose beads, by Roche (1 mentions)
X ray film, by Kodak (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Mouse anti gfp antibody, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence detection, by Cytiva (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Tnt reticulocyte lysate system, by Promega (1 mentions)
Semi dry blotting, by Bio-Rad (1 mentions)
2 protocols using protein a beads
1
Purification and Identification of Protein Complexes in S. solfataricus
2
Co-immunoprecipitation of RNF217 and HAX1
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Radioactively labeled [35S]-proteins were produced using the TNT-reticulocyte-lysate system (Promega, Mannheim, Germany). RNF217 was expressed from the pGBKT7 vector to give rise to a HA-tagged protein, HAX1 was expressed from the pGADT7 vector to give rise to a myc-tagged protein. The in vitro translated proteins were mixed 1:1 and immunoprecipitated with an anti-HA (RNF217) or an anti-myc antibody (HAX1) together with protein A beads (Clontech). Proteins were separated by SDS-PAGE, the gel was fixed and incubated with fluorographic amplification reagent (Amersham) before being exposed to an X-ray film (Kodak, Stuttgart, Germany).
pCFP-RNF217 and pYFP-HAX1 were co-transfected into HEK293T cells and the cells were lysed 24 h later. HAX1 was immunoprecipitated with a mouse anti-HAX1 antibody (Clontech) or control polyclonal mouse IgG (Santa Cruz, Heidelberg, Germany) and protein G-agarose beads (Roche, Mannheim, Germany). Proteins were separated by SDS-PAGE, blotted on nitrocellulose membranes (BioRad, Munich, Germany) by semi-dry blotting (BioRad), and CFP-RNF217 was detected using an mouse anti-GFP antibody (Molecular Probes, Darmstadt, Germany) with an HRP-conjugated secondary anti-mouse antibody (Santa Cruz) and enhanced chemiluminescence detection (Amersham).
pCFP-RNF217 and pYFP-HAX1 were co-transfected into HEK293T cells and the cells were lysed 24 h later. HAX1 was immunoprecipitated with a mouse anti-HAX1 antibody (Clontech) or control polyclonal mouse IgG (Santa Cruz, Heidelberg, Germany) and protein G-agarose beads (Roche, Mannheim, Germany). Proteins were separated by SDS-PAGE, blotted on nitrocellulose membranes (BioRad, Munich, Germany) by semi-dry blotting (BioRad), and CFP-RNF217 was detected using an mouse anti-GFP antibody (Molecular Probes, Darmstadt, Germany) with an HRP-conjugated secondary anti-mouse antibody (Santa Cruz) and enhanced chemiluminescence detection (Amersham).
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