To study the interaction between AR and ATF3, we immunoprecipitated protein extract with polyclonal AR or ATF3 antibody followed by ATF3 or AR immunoblot analysis. Briefly, edelfosine (5 μM) treated LNCaP cell lysates (200 μg) were incubated each with 1 μg (AR/N-20 or ATF3/H-90, Santa Cruz, Dallas, TX) of AR or ATF3 antibody overnight followed by incubation with protein G-Sepharose beads (Life Technologies, Grand Island, NY) at 4°C for 1 h. Immunocomplexes were washed three times with lysis buffer and were denatured by treatment with SDS sample-loading buffer at 100 °C for 10 minutes followed by immunoblotting with ATF3 or AR specific antibodies. Proteins were visualized using an enhanced chemiluminescence system (GE Healthcare Bio-science, Piscataway, NJ).
Protein g sepharose beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protein G Sepharose beads are a solid-phase affinity chromatography medium used for the purification of immunoglobulins (Ig) and other proteins that bind to Protein G. Protein G is a bacterial cell wall protein that exhibits a high affinity for the Fc region of most immunoglobulin classes and subclasses, allowing for the efficient capture and isolation of Ig from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protein g sepharose beads, by Merck Group (3 mentions)
Hitrap igm column, by GE Healthcare (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Ab10530, by Abcam (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Ar n 20, by Santa Cruz Biotechnology (1 mentions)
Sepharose 4b beads, by Merck Group (1 mentions)
Easytag express35s protein labeling mix, by PerkinElmer (1 mentions)
Typhoon trio imager, by GE Healthcare (1 mentions)
Cdp star western blot chemiluminescence reagent, by PerkinElmer (1 mentions)
Sigmafast edta free protease inhibitor cocktail, by Merck Group (1 mentions)
Dimethyl pimelimidate, by Merck Group (1 mentions)
Micro bio spin chromatography column, by Bio-Rad (1 mentions)
Tla120.2 rotor, by Beckman Coulter (1 mentions)
56 protocols using protein g sepharose beads
1
Interaction of AR and ATF3 Proteins
2
Metabolic Labeling and OPA1 Isoform Analysis
3
Vimentin Immunoprecipitation and O-GlcNAc Detection
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MEFs were lysed in the buffer containing 50 mm Tris-Cl (pH 7.5), 150 mm NaCl, 50 mm sodium pyrophosphate, 50 mm NaF, 50 mm sodium β-glycerophosphate, 1 mm Na3VO4, 2 mm EDTA, 0.5% deoxycholate, 0.1% SDS, and 1% Nonidet P-40. After the centrifugation (17,400 × g), each supernatant was incubated with 2 μl of goat anti-vimentin (antisera) (34 (link)) for 30 min, followed by an additional 30-min incubation with 10 μl of protein G-Sepharose beads (Life Technologies, Inc.). All the above procedures were performed at 4 °C. Each immunoprecipitate was immunoblotted with rabbit anti-vimentin or mouse anti-O-GlcNAc (clone CTD110.6, Cell Signaling Technology) monoclonal antibodies.
4
Immunoprecipitation and Western Blotting of Flt3 and Fes
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AML cells (2.5 x 106 in 5 mL) were cultured in the presence of kinase inhibitors or DMSO alone for 6 to 16 hours prior to lysis by sonication in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate) supplemented with 1 mM sodium orthovanadate, 1 mM sodium fluoride, 5 units/ml Benzonase (Novagen), and SigmaFAST EDTA-free protease inhibitor cocktail (Sigma). Flt3 and Fes were immunoprecipitated from 1 mg of cell lysate with 2 μg of antibody and 25 μL of protein G-Sepharose beads (Life Technologies) overnight at 4°C. Immunoprecipitates were washed three times by resuspension in 1.0 ml of RIPA buffer. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with the indicated antibodies followed by alkaline phosphatase-linked secondary antibodies. CDP-Star Western Blot Chemiluminescence Reagent (Perkin-Elmer) was used for detection.
5
Gag-EGFP Protein Immunoprecipitation
6
Isolation and Immunoprecipitation of Hck and Lyn
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Stimulated BMDMs were lysed in 1% Lauryl Maltoside Lysis Buffer containing 150 mM NaCl, 0.01% sodium azide, and 10 mM Tris, pH 7.6 with 2 mM NaVO4, 0.01 mg/ml Aprotinin, 0.01 M NaF, 0.01 mg/ml Leupeptin, 0.01 mg/ml Pepstatin A, 2 mM phenylmethanesulfonyl fluoride (PMSF), and 0.4 mM EDTA. After scraping the plates, cells and detergent were incubated 30 min on ice. The lysate was then cleared by ultracentrifugation for 15 min at 50,000 rpm at 4°C in a Beckman (Pasadena, CA) TLA120.2 rotor. The lysates were precleared for 30 min at 4°C with Protein G Sepharose beads (Life Technologies, Carlsbad, CA) and normal rabbit or goat serum as appropriate (Jackson ImmunoResearch, West Grove, PA). Protein G-Sepharose beads were covalently conjugated to Lyn or Hck antibodies (see table below) using dimethyl pimelimidate (Sigma). Antibody-bound beads were added to the lysate and mixed 1.5 hr at 4°C to immunoprecipitate Hck or Lyn. Finally, the samples were applied to micro bio-spin chromatography columns (Bio-Rad, Hercules, CA), washed, and eluted with SDS Sample Buffer.
7
ChIP-qPCR Analysis of Lipin1 Binding
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Livers (300 mg) were lysed in a buffer containing 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP-40, and protease inhibitors. The samples were crosslinked using 1% formaldehyde for 10 min at room temperature, and were then prepared for ChIP as described previously22 (link). The crosslinked samples were incubated with anti-lipin1 antibodies (R&D Systems) and protein G-Sepharose beads (Life Technologies Japan; Tokyo, Japan) overnight at 4 °C. The beads were then washed several times, and bound proteins were eluted in elution buffer (1% sodium dodecyl sulfate in 0.1 M NaHCO3). The elutes were treated with pronase (1 mg/mL; Roche) for 2 h at 42 °C, and were then were incubated at 65 °C overnight to remove the crosslinks. The samples were further purified using nucleospin extract II (TaKaRa; Tokyo, Japan). qPCR was performed using the primers listed in Table S1 in the supplemental material.
8
Immunoprecipitation and Western Blot Protocol
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Cells were lysed in IP lysis buffer containing 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, EDTA-free protease inhibitor cocktail (Roche), and 1 mM phenylmethylsulfonyl fluoride. Cell lysates were incubated overnight at 4°C with anti-Flag M2, anti-HA, or anti–c-Myc agarose affinity gel antibody (Sigma-Aldrich) or anti-IRF7 or anti-Pin1 antibodies and protein G Sepharose beads (Thermo Fisher Scientific). Beads were washed three times and eluted with 2× SDS loading buffer. For immunoblotting, proteins were resolved by SDS–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane.
9
Isolation and Characterization of Insulin-Specific Immunoglobulins
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Serum of immunized mice was collected immediately after euthanization. Removal of antigen bound to antibodies was achieved by repeated freeze-thaw cycles of the serum and pH-shift during elution (23 (link)). Protein G sepharose beads (ThermoFisher) were used according to the manufacturers protocol and dialyzed overnight in 10 times sample volume in 1x PBS to isolate IgG. For IgM, HiTrap IgM columns (GE Healthcare, Sigma-Aldrich) were used according to the manufacturers protocol and dialyzed overnight in 10 times sample volume 1 x PBS. Quality-control of the isolated immunoglobulins was done via SDS-PAGE and Coomassie-staining and the concentration of insulin-specific immunoglobulins determined via ELISA.
10
Western Blot and Co-Immunoprecipitation Protocols
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For WB, briefly, primary antibodies were purchased from Cell signalling Technology, Affinity Biosciences (OH, USA), Abclonal, Proteintech, or MBL (Janpan). Anti-DACH1 antibody (Proteintech Cat. 10914-1-AP, 1:1000), anti-SMAD4 antibody (CST, Cat. #46535, 1:1000), anti-Phospho-Smad4 (Thr276)[Thr277] antibody (Affinity Biosciences, Cat. AF8316, 1:1000), anti-LGR5 antibody (Abclonal, Cat. A10545, 1:1000), anti-NICD (Cleaved-Notch 1 (Val1744) antibody, Affinity Biosciences, Cat. AF5307, 1:1000), anti-GAPDH antibody (Proteintech, Cat. 10494-1-AP, 1:5000) and anti-FLAG (Anti-DDDDK-tag) antibody (MBL, M185-3 L, 1:5000) were incubated with the membrane respectively, followed by washing for 3 times with TBST and incubation with according HRP-conjugated secondary antibodies. After washing off the unbound secondary antibodies with TBST, the bands were detected using LAS 4000 (GE, MA, USA). For CoIP, total cell lysates were incubated with anti-DACH1 or anti-SMAD4 antibody followed by incubation with protein G-Sepharose beads (Thermo Fisher Scientific). The beads were washed three times with lysis buffer. The immunoprecipitates were eluted by boiling the beads for 5 min in SDS loading buffer and subjected to WB as described above.
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