Cells were plated with culture medium supplemented with 10% complete serum in six-well plates at a density of 300,000 per well. After 24 h, cells were treated for 48 h as indicated and then lysed in a 50 mM Tris–HCl buffer (pH 7.4) containing 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and fresh Protease Inhibitors Cocktail (1%; Sigma). After homogenization by passing 10 times through a 1 cm3 microfine insulin syringe, lysates were cleared by centrifugation at 14,000 rpm for 5 min in a bench-top microfuge, and 15% of the lysate solution was set aside and used as input. The remaining lysate was immunoprecipitated with approximately 3 μg of the specified antibody and 30 μl protein-G beads (Amersham Biosciences, Freiburg, Germany) and washed three times for 5 min each with the same buffer, followed by centrifugation at 4,000 rpm for 1 min. Thirty microliters of the immunoprecipitate/bead suspension were mixed with 20 μl of 2.5× “sample buffer” (50% Glycerol, 10% SDS, 1 M Tris–HCl pH 6.8, 25% beta ß-Mercaptoethanol, 0.5% Bromopenol blue) and boiled for 5 min. Input and immunoprecipitates were subjected to Western blot analysis with either anti-FLAG antibodies (Sigma) to detect FLAG-RUNX2 or anti-GR antibodies cat. no. sc-1004 (M20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Protein g beads
Manufactured by Cytiva
Sourced in United States, United Kingdom
Protein G beads are a type of chromatography resin used for the purification of antibodies. Protein G is a bacterial cell wall protein that binds to the Fc region of immunoglobulins with high affinity. The beads are made of agarose or other suitable matrix material and are functionalized with covalently coupled Protein G. They can be used in various antibody-based applications, such as immunoaffinity chromatography, immunoprecipitation, and Western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitors cocktail, by Merck Group (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti erα mc20, by Santa Cruz Biotechnology (1 mentions)
Anti striatin, by BD (1 mentions)
Phospho ampk, by Cell Signaling Technology (1 mentions)
Proteome profiler antibody array, by R&D Systems (1 mentions)
Pp2ac, by Merck Group (1 mentions)
Ecl prime, by Cytiva (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Protease inhibitor, by Cytiva (1 mentions)
Myd88, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Silver stain plus kit, by Bio-Rad (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Protein lobind tube, by Eppendorf (1 mentions)
10 protocols using protein g beads
1
Immunoprecipitation and Western Blot Analysis
2
Coimmunoprecipitation and Western Blot Analyses
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Tissue proteins were extracted in IP Lysis Buffer (Thermo Fisher Scientific) mixed with cOmplete Protease Inhibitor Cocktail (Roche), and lysates were incubated overnight at 4°C with 5 μg anti-ERα (MC20; Santa Cruz Biotechnology) or anti-striatin (BD Bioscience) antibody. Next, the lysates were incubated with protein G beads (Amersham Biosciences) for 2 h at 4°C, and the pellets obtained after centrifugation were washed five times and analyzed by immunoblotting. Proteins were resolved by dodecyl sulfate-PAGE, transferred to polyvinylidene fluoride membranes, and probed with the appropriate primary antibodies, including AMPK, GAPDH (Santa Cruz Biotechnology), PP2Ac (Millipore), phospho (p)-AMPK, p-Akt, total Akt (Cell Signaling Technology), and α-tubulin (EMD). Membranes were then incubated with the appropriate secondary antibodies and developed using ECL Prime (Amersham Biosciences). The Proteome Profiler Antibody Array (R&D Systems) was used for phospho-kinase analysis, according to the manufacturer’s instructions.
3
Immunoprecipitation of MyD88 Protein
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Sample lysates were prepared in lysis buffer (20 mM Tris, 10% Glycerol, 5 nM MgCl2, 0.1% Tween 20, 0.3 M KCl, 1/1000 2-mercaptoethanol) containing phosphatase inhibitors and protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Total protein (500 µg) was mixed with a primary antibody against MyD88 (Cell Signaling Technology, Danvers, MA, USA) at 4℃ overnight. Protein-G beads (Amersham Biosciences, Sunnyvale, CA, USA) were added and incubated at 4℃ for 2~3 h. The precipitates were washed three times with lysis buffer containing phosphatase and protease inhibitors as described above. The precipitates were fractionated on a SDS-PAGE gel followed by the immunoblotting procedure described above.
4
Tyrosine Phosphorylated Podocyte Proteins Isolation
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Tyrosine phosphorylated podocyte proteins were isolated using an antiphosphotyrosine antibody bound to protein G beads (Amersham, UK). Cell lysates were clarified by centrifugation, then added to the washed beads, along with protease and phosphatase inhibitors, and left to bind overnight.
Following a brief spin, the supernatant was removed and the beads washed in ice-cold lysis buffer. The pellet was resuspended in Laemmli sample buffer containing 2-mercaptoethanol. Bound proteins were released by boiling and run on an SDS-polyacrylamide gel. Protein bands were visualised by silver staining and were carried out using a Silver Stain Plus Kit (Bio-Rad, UK), as per the manufacturer's instructions.
Following a brief spin, the supernatant was removed and the beads washed in ice-cold lysis buffer. The pellet was resuspended in Laemmli sample buffer containing 2-mercaptoethanol. Bound proteins were released by boiling and run on an SDS-polyacrylamide gel. Protein bands were visualised by silver staining and were carried out using a Silver Stain Plus Kit (Bio-Rad, UK), as per the manufacturer's instructions.
5
Immunoprecipitation and GST Pull-down Assay of Cep169 and CDK5RAP2
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293T cells were transfected with EGFP-Cep169 and FLAG-CDK5RAP2 and extracted by extraction buffer (20 mM Tris-HCl at pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.2% Nonidet P-40, 10% glycerol, 1 mM NaF, 1 mM Na3VO4, 20 mM β-glycerophosphate, 10 mM β-mercaptoethanol, 0.2 mM PMSF, Protease Inhibitor Cocktail [Nacarai Tesque]) by two rounds of freezing and thawing as described previously [16 (link)]. An equal amount of each lysates were incubated with anti-FLAG antibody or rabbit IgG coupled to protein G beads (Amersham) for 1 h at 4°C. After washing the beads three times with extraction buffer, the immune complexes were analyzed by Immunoblotting. For in vitro GST pull-down assay, the recombinant protein of GST-EB1 was expressed in Escherichia coli and purified by affinity chromatography on glutathione-sepharose 4B (GE Healthcare). The recombinant protein of GST-EB1 and glutathione sepharose-beads were mixed with cell extracts from 293T transiently expressed with FLAG-Cep169 wild type or SxAA mutant. Consequent to incubation for 12 h at 4°C under continuous rotation beads were washed in lysis buffer three times. Proteins were eluted in Laemmli buffer, separated by SDS-PAGE and analyzed with immunoblotting (IB) using anti-FLAG antibody or Coomassie Brilliant Blue (CBB) staining, respectively.
6
Immunoprecipitation and Analysis of Coenzyme Q9
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Two types of Psap polyclonal antibodies were used. One antibody was obtained from Dr. Matsuda (Tokai University), and the other was purchased from Santa Cruz Biotechnology (Dallas, TX). The antibodies were generated by immunizing rabbits with synthetic oligopeptides corresponding to 178-LYPQDHPRSQPQPKAN-193, 299-EMMDPYEQNLVQAH-312, 413-KEPTPPKQPAQPKQSALP-430,(13 ) and 301-MDPYEQNLVQAHNVILCQTCQFVMNKFSELIVNNATEELLVKGLSNACALLPDPARTKCQEVVGTFGPSLLDIFIHEVNPSSLCG-385 (Santa Cruz, technical note sc-32876) of the 557-amino acid mouse Psap (UniProt: Q61207), respectively.
Samples were placed in protein LoBind tubes (Eppendorf, Hamburg, Germany) and incubated with 5 μg of anti-Psap IgG or normal rabbit IgG for 1 h at room temperature. We tested two different types of anti-Psap IgG. After incubation, 10 μl of protein G beads (Amersham Biosciences, Buckinghamshire, UK) were added to the samples, followed by further incubation for 1 h. The beads were then washed three times with PBS. CoQ9 was extracted from the beads using 2-propanol and analyzed by HPLC-ECD. The beads were mixed with SDS-PAGE sample buffer, and Psap was analyzed by Western blotting.
Samples were placed in protein LoBind tubes (Eppendorf, Hamburg, Germany) and incubated with 5 μg of anti-Psap IgG or normal rabbit IgG for 1 h at room temperature. We tested two different types of anti-Psap IgG. After incubation, 10 μl of protein G beads (Amersham Biosciences, Buckinghamshire, UK) were added to the samples, followed by further incubation for 1 h. The beads were then washed three times with PBS. CoQ9 was extracted from the beads using 2-propanol and analyzed by HPLC-ECD. The beads were mixed with SDS-PAGE sample buffer, and Psap was analyzed by Western blotting.
7
Immunoprecipitation of Protein Complexes
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HEK293 cells were incubated for 24 hr with DNA encoding various proteins in the presence of Genejuice. Immunoprecipitation was initiated by incubation of lysates for 2 hr with protein A/G sepharose beads (Amersham) plus control antibodies. Precleared lysates were then incubated at 4°C for at least 2 hr with various antibodies and protein G beads (Amersham) prior to separation by SDS- PAGE and visualization with an enhanced chemiluminescence system (Li-Cor).
8
Generation of monoclonal antibodies against tBc48
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SP2/0 myeloma cells were cultured a week prior to the organ harvesting. Spleen of the immunised mice was removed and spleen cells were suspended in Roswell Memorial Institute (RPMI)-1640 medium (Gibco-BRL Life Technologies, Grand Island, NY, USA). Spleenocytes were fused with SP2/0 myeloma cells using 50% polyethylene glycol-4000 (PEG4000). The resulting hybridoma cells were cultured in RPMI-1640 medium containing HAT (hypoxanthine-aminopterin-thymidine) and HT (aminopterin-thymidine) (Sigma) for 10-14 days. The hybridoma cells secreting antibody against tBc48 were screened by ELISA and then subcloned 4-5 times by limiting dilution assay. Secreting antibody was finally confirmed by IFA of tBc48 transfected 293T cells.
Ascites fluid containing mAb was prepared by injecting 5 × 10 5 selected hybridoma cells into the abdominal cavity of each mouse. One week prior to the culture of ascitic fluid, mice were injected with sterile liquid paraffin. mAbs collected from ascitic fluid were purified using protein-G beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Immunoglobin subtypes of the mAbs were identified with Southern Biotechnology Associates Clonotyping TM Horseradish peroxidase system (HRP) according to the manufacturer's protocols (Southern Biotechnology Associates, Birmingham, AL, USA).
Ascites fluid containing mAb was prepared by injecting 5 × 10 5 selected hybridoma cells into the abdominal cavity of each mouse. One week prior to the culture of ascitic fluid, mice were injected with sterile liquid paraffin. mAbs collected from ascitic fluid were purified using protein-G beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Immunoglobin subtypes of the mAbs were identified with Southern Biotechnology Associates Clonotyping TM Horseradish peroxidase system (HRP) according to the manufacturer's protocols (Southern Biotechnology Associates, Birmingham, AL, USA).
9
Insulin Receptor Autoantibody Detection
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The principle of IRAb detection by IP assay is shown in Figure 2A
10
Immunoprecipitation of FLAG-tagged Proteins
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Cells were seeded at 4 x 106 cells per plate and induced with 1 μg/mL doxycycline for 24 hr. The cells were harvested by first washing with cold 1X PBS, scraping, and collecting the cells in 1 mL 1X PBS. The cells were spun down at 500 x g for 5 min at 4ºC, and lysed in 1X IP lysis buffer (50 mM Tris pH 7.6, 100 mM KCl, 2 mM EDTA, 0.1% IGEPAL, 10% glycerol). The lysate was flash frozen on dry ice for 10 min and then allowed to thaw at room temperature. The lysate was then sonicated 3X for 10 sec at 1.5 amp. After sonication, the lysate was digested with 1 U/μL MNase and the buffer supplemented with 5 mM CalCl2. This was left to incubate overnight with rocking at 4ºC. Four milligrams of protein was incubated with anti-FLAG (1 μg/mg lysate) overnight at 4ºC, rocking. The following day, magnetic Protein G beads (Cytiva, 28967070) were washed three times in 1X IP lysis buffer and the lysate and antibody mix was added onto the beads and incubated at 4ºC for 4 hr with rocking. The supernatant was removed, and the beads were washed three times in 1X IP lysis buffer. Proteins were eluted with 40 μL 1X Laemmli (2% SDS, 0.1% bromophenol blue, 10% glycerol, 62.5 mM Tris pH 6.8, 100 mM DTT) and boiled at 95ºC for 15 min.
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