RD sarcoma cells were treated with Bazedoxifene 15μM for 16 hours. Cell lysates were prepared by adding lysis buffer (Cell Signaling Technology, Danvers, MA) and were pre-cleared using protein A/G agarose (Pierce Biotechnology, Rockford, IL), incubating with gentle mixing at 4°C for 2 h. GP130 was immunoprecipitated by incubating cell lysates with anti-GP130 antibody (EMD Millipore Corporation, Temecula, CA) overnight at 4°C. Protein agarose slurry was added and further incubated for 2 h at 4°C. At the end of incubation, protein A/G agarose was washed three times with IP buffer (Thermo, #28379) and proteins bound to GP130 were collected by boiling the samples in 5x loading buffer. The supernant was then separated by 10% SDS-PAGE and subjected to western blot analysis with 1:1000 dilution of primary antibodies and 1:10000 horseradish peroxidase-conjugated secondary antibodies. Antibodies against IL-6R, STAT3, GP130, and JAK1 were used for western blotting.
Protein a g agarose
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
Protein A/G agarose is a solid support matrix composed of agarose beads with covalently coupled protein A and protein G. It is designed for the purification of antibodies and immunoglobulins from various sample sources.
Automatically generated - may contain errors
Lab products found in correlation
Ip buffer, by Thermo Fisher Scientific (5 mentions)
Talon purification kit, by Takara Bio (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Anti gp130 antibody, by Merck Group (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Captureselect iga affinity matrix, by Thermo Fisher Scientific (3 mentions)
Anti ha, by Merck Group (3 mentions)
Anti mouse igg hrp linked secondary antibody, by Cell Signaling Technology (3 mentions)
Complete protease inhibitor, by Merck Group (3 mentions)
Pet28a expression vector, by Merck Group (2 mentions)
Anti v5 hrp antibody for tag detection, by Thermo Fisher Scientific (2 mentions)
Igg elution buffer, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer 2, by Thermo Fisher Scientific (2 mentions)
Monoclonal mouse anti ha agarose beads, by Merck Group (2 mentions)
Monoclonal mouse anti myc antibodies, by Takara Bio (2 mentions)
Protease inhibitor cocktail, by GenDEPOT (2 mentions)
E412 01, by Vazyme (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Anti ha agarose, by Merck Group (2 mentions)
127 protocols using protein a g agarose
1
Investigating GP130 Signaling Pathway in RD Sarcoma Cells
2
Immunoprecipitation and Mass Spectrometry of ASCL1
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Nuclear fractions from ∼1 × 106 NCI-H2107 cells were prepared using the NE-PER kit (ThermoFisher Scientific 78,835) as per the manufacturer's instructions and incubated with 10 μg primary antibodies overnight at 4°C followed by 90 min incubation with 35 μL protein A/G agarose plus (50% slurry) (ThermoFisher Scientific, 20,421). Immune pellets were washed 5 times in PBS containing 1% Triton X-100 and analyzed by SDS-PAGE followed by either immunoblotting or mass spectrometry (LC/MS-MS). For LC/MS-MS, 6 immunoprecipitations were pooled. Antibodies used for immunoprecipitations were mouse anti-ASCL1 antibodies (BD-Biosciences 556604) and normal mouse IgG (Santa Cruz Biotechnology sc-2025). For Figure 3 B, a guinea pig anti-ASCL1 antibody was used (TX518 (Kim et al., 2008 (link))). Nuclear extracts of NCI-H524 cells, which do not express ASCL1 were used as negative control. For NCI-H889, NCI-H2171, and NCI-H345, cells were lysed by incubation in RIPA buffer for 30 min on ice. Protein concentration of lysate was determined by BCA assay kit (Pierce 23,225) and 200 μg total lysate was incubated overnight at 4°C on an inverter with 35 μL protein A/G agarose plus (50% slurry) (ThermoFisher Scientific, 20,421) pre-loaded with guinea pig anti-ASCL1 antibody (TX518). Immune pellets were washed 5 times in PBS containing 1% Triton X-100 and analyzed by SDS-PAGE followed by immunoblotting.
3
Bazedoxifene Modulates IL-6 Signaling
4
Immunoprecipitation of RAW264.7 Cell Lysates
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Immunoprecipitation was performed as described previously (21 (link)). Briefly, RAW264.7 lysates were prepared in immunoprecipitation lysis buffer (20 mmol/L Tris-Cl, pH 8.0, 10 mmol/L NaCl, 1 mmol/L EDTA, 0.1% NP-40) containing a protease inhibitor cocktail (Sigma). Two micrograms of cell extracts were precleared with 50 µl of protein A/G-agarose (Thermo Fisher) at 4°C for 2 h, and the supernatant was incubated with the corresponding antibodies with gentle shaking at 4°C overnight, followed by the addition of 20 µl of protein A/G-agarose for another 1 h. The beads were washed and then resuspended in 30 µl of loading buffer and boiled for 5 min, followed by Western blot detection.
5
Protein Immunoprecipitation and Western Blot Analysis
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Cells were cultured in normoxia or hypoxia for at least 72 h and were lysed at 4 °C in NP-40 buffer containing protease inhibitor cocktail, 1 mM Na3VO4, 2 mM sodium fluoride and 1 mM phenylmethylsulfonyl fluoride. One to three milligrams of protein was immunoprecipitated at 4 °C overnight using either 30 μg of monoclonal anti-huCAIX antibody or 15 μg of monoclonal anti-MMP14 antibody covalently linked to CNBr-activated Sepharose 4B as described previously.66 (link) In the case of co-IP with integrins, 1 mg of the cell lysate was immunoprecipitated at 4 °C overnight using 10 μg of monoclonal mouse anti- integrins β1and α2 and then incubated for 1 h at 4 °C with Protein A/G Agarose and UltraLink Resin (Cat no. 53132, ThermoFisher Scientific, Burlington, ON, Canada). The resin was extensively washed with NP-40 buffer pH 8.0 with 1% Tween-20, resuspended in sample buffer and boiled at 100 °C for 10 min under non-reducing conditions. The proteins in the supernatant were separated from the resin by centrifugation using a polypropylene spin column (Bio-Rad, Mississauga, Ontario, USA) for 1 min, at 4000 rpm. β-mercaptoethanol was added to the supernatant and eluates were boiled again. All samples were loaded on SDS–PAGE gels and western blots were performed as described above.
6
Recombinant ZIKV NS1 Protein Production
7
Immunoprecipitation of Myc-tagged Proteins
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Coverslips were prepared in advance and placed in six-well plates. C2C12 myoblasts were seeded into the plates and fixed in 4% paraformaldehyde. The cell membranes were permeabilized in 0.1% (wt/vol) Triton X-100, and the cells were blocked in PBS (phosphate buffered saline) containing 3% BSA (bovine serum albumin) and stained with antibodies in PBS containing 3% BSA. Primary antibodies against Suv39h1 (Abcam, Cambridge, Cambridgeshire, UK), MEF2C (Abcam), HP1α (Abcam), and MHC (Abcam) were diluted at 1:500. Secondary fluorochrome-conjugated antibodies (Boster, Wuhan, China) were diluted at 1:64. DAPI (diamidino-phenyl-indole) was used as the nuclear counterstain.
Cells were washed three times in PBS, scraped from 10 mm2 culture dishes and suspended in NP40 cell lysis buffer. Cell lysates were incubated on ice by repeated pipetting for 30 min and centrifugation in a microfuge for 10 min at 13,000× g, at 4 °C. For co-immunoprecipitations, antibodies (2 μg anti-Myc, MilliporeSigma, Billerica, MA, USA) were incubated with 500 μL of cell extract supernatant overnight at 4 °C. Then, this immune complex was immobilized on 20 μL protein A/G agarose (Thermo Fisher Scientific) and incubated for 2 h at 4 °C. The immunoprecipitates were washed thrice in wash buffer, eluted in 20 μL elution buffer and analyzed by Western blot.
Cells were washed three times in PBS, scraped from 10 mm2 culture dishes and suspended in NP40 cell lysis buffer. Cell lysates were incubated on ice by repeated pipetting for 30 min and centrifugation in a microfuge for 10 min at 13,000× g, at 4 °C. For co-immunoprecipitations, antibodies (2 μg anti-Myc, MilliporeSigma, Billerica, MA, USA) were incubated with 500 μL of cell extract supernatant overnight at 4 °C. Then, this immune complex was immobilized on 20 μL protein A/G agarose (Thermo Fisher Scientific) and incubated for 2 h at 4 °C. The immunoprecipitates were washed thrice in wash buffer, eluted in 20 μL elution buffer and analyzed by Western blot.
8
Recombinant Protein Expression and Purification
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HEK293T/17 cells were transiently transfected with different protein expression plasmids using Lipofectamine 3000 (Life Technologies). Then, 6 h posttransfection, the cells were washed with phosphate-buffered saline (PBS) twice and cultured in a fresh 293T FreeStyle expression medium (Life Technologies) at 37°C in a humidified 5% CO2 incubator. The supernatant was collected at 48 h posttransfection and centrifuged at 4,000 × g for 10 min at 4°C. The clarified supernatant was purified with protein A/G agarose (Thermo Scientific) and eluted with IgG elution buffer (Thermo Scientific). The HE-Stag proteins of BCoV-Mebus and PToV-Markelo were expressed and purified as previously described (60 (link)). The purified proteins were buffered with PBS, quantified using a Qubit 2 fluorometer (Thermo Scientific), and then aliquoted and stored at −80°C for further use.
9
Immunoprecipitation of Giardia Proteins
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Giardia cells carrying pGlPLK-HA.neo and pGlKin-13-Myc.pac were harvested, washed 3 times with ice-cold PBS, and lysed with lysis buffer (10 mM Tris-Cl, 5 mM EDTA, 130 mM NaCl, and 1% Triton X-100, pH 7.4) containing protease inhibitor cocktail (GenDEPOT, Katy, Taxas, USA). Lysates precleared using Protein A/G Agarose (Thermo Fisher Scientific) for 1 h at 4°C were incubated with monoclonal mouse anti-HA agarose beads (Sigma-Aldrich) or monoclonal mouse anti-Myc antibodies (Clontech, Mountain View, California, USA) at 4°C overnight. Beads were washed with immunoprecipitation washing buffer (50 mM Tris-Cl, 150 mM NaCl, and 1% Triton X-100, pH 7.4) and resuspended in 2×SDS sample buffer. The samples were analyzed by Western blotting using anti-HA or anti-Myc antibodies.
10
L1CD Protein Immunoprecipitation and Immunoblot
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