The treated MARC-145 or HEK-293FT cells in six-well plates were harvested and lysed in Nonidet P-40 buffer (0.5% Nonidet P-40, 150 mM NaCl, 50 mM Tris⋅HCl, pH 8.0) containing protease inhibitors (Sigma, #P8340). After being clarified by centrifugation at 12,000 rpm for 20 min, the supernatants were precleared with protein A/G Sepharose beads (Santa Cruz, #sc-2003) before being incubated with 3 to 5 μL indicated antibodies and protein A&G Sepharose beads overnight at 4 °C. The beads were washed and then subject to SDS/PAGE and Western blot analysis.
Protein a g sepharose beads
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Protein A/G sepharose beads are agarose beads coupled with recombinant Protein A and Protein G. These beads are used for the purification of antibodies from various sample sources.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor, by Roche (3 mentions)
Phosphatase inhibitor, by Roche (3 mentions)
Supersignal west pico, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by AnaSpec (3 mentions)
M per lysis buffer, by Thermo Fisher Scientific (3 mentions)
Clean blot ip detection kit hrp, by Thermo Fisher Scientific (2 mentions)
Ip buffer, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Immunoprecipitation lysis buffer, by Beyotime (2 mentions)
Lysis buffer, by Beyotime (2 mentions)
Control mouse igg, by Santa Cruz Biotechnology (2 mentions)
Anti maf, by Novus Biologicals (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Bioruptor generated soluble chromatin, by Cosmo Bio (2 mentions)
Sample buffer, by Bio-Rad (2 mentions)
Halt protease and phosphatase inhibitors cocktail, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
120 protocols using protein a g sepharose beads
1
Protein Immunoprecipitation and Western Blot
2
PRRSV nsp2 Immunoprecipitation Assay
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MARC-145 cells in six-well plates were infected with PRRSV strain JXwn06 at an MOI of 0.1. At 24 hpi, the cells were harvested and lysed in NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0) containing protease inhibitor (Sigma, #P8340). After clarifying by centrifugation at 12, 000 rpm for 20 min, the supernatants were precleared with protein A/G sepharose beads (Santa Cruz, #sc-2003) and then incubated with 5 μL mouse anti-nsp2 MAb (mouse isotype antibody as a negative controls) and 25 μL protein A&G sepharose beads overnight at 4°C with gentle rotation. The beads were washed five times with the NP-40 lysis buffer, and the proteins bounded to the beads were separated by SDS-PAGE, followed by western blot analysis.
3
Immunoprecipitation and Western Blot Analysis
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Total cell lysates were collected from the HEK 293 cells transfected with different sets of expression vectors, and then pre-cleared with 30 μl protein A/G-Sepharose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) to remove nonspecific proteins. Following 1 h of incubation, centrifugation was conducted at 600 × g for 5 min, to separate the cell lysates from the beads. The pre-cleared supernatants were then incubated for 3 h with 2 μg mouse monoclonal anti-human c-myc or synthetic flag antibodies (sc-3777551 and sc-807, respectively; Santa Cruz Biotechnology, Inc.), and then incubated for 12 h with 30 μl protein A/G-Sepharose beads at 4°C under gentle rotation. The protein-bead complexes were precipitated by centrifugation at 600 × g for 5 min, washed five times with washing buffer [1:1 mixture of lysis buffer and phosphate-buffered saline (PBS)] and mixed with 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Subsequent to boiling for 5 min, the immunoprecipitated samples were resolved on SDS polyacrylamide gel and subjected to western blot analysis.
4
Immunoprecipitation and Western Blot Analysis of Viral Proteins
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Cells were lysed with RIPA buffer (Cell Signaling) supplemented with 0.1 M phenylmethyl sulfonyl fluoride (PMSF), protease inhibitor mixture, and phosphatase inhibitors 2 & 3 (Sigma)). Protein concentrations for western blotting and immunoprecipitation were determined using the Bio-Rad protein assay reagent and a Beckman Coulter spectrophotometer. Immunoprecipitation experiments were conducted as described previously [63 (link)]. Briefly, 500 μg of protein was used for each immunoprecipitation and precleared with 50 μl protein A/G sepharose beads (Santa Cruz) for 6 h. Antibody (2 μg) was incubated with 20 μl protein A/G sepharose beads for 6 h to overnight. The immune-complexes were washed 3 times with RIPA buffer before being resolved using 2× SDS-PAGE loading buffer (sigma) and separated on a 4–20% Tris-HCl gradient SDS-PAGE gel (BioRad). The signal was detected using the Odyssey Infrared Imaging System (Li-Cor Biosciences). The following antibodies were used: Actin (sc-1616), Zta (Argene 11–007), Rta (Argene 11–008), BMRF1 (EBV-018-48,180), BGLF4 (Argent AP8057b), VCA (Argene 11–019), LMP1 (BD 559898) and GAPDH (Cell Signaling 2118 L).
5
Immunoprecipitation and Western Blotting Protocol
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Cells (as indicated in each experiment) were harvested and homogenized in the lysis buffer (Beyotime, P0013) supplemented with protease inhibitor cocktail (Roche, 04693132001) at 4 ºC for 30 min. Protein concentration was measured using a Pierce BCA Protein Assay kit (Thermo Fish Scientific, 23227). Cell lysates were pre-cleared with 20 μL protein A/G-Sepharose beads (Santa Cruz, sc-2003). The pre-cleared cell lysates were incubated with 2 μg YAP1 (Mouse monoclonal, Santa Cruz, sc-101199), CLP36 (Mouse monoclonal, Santa cruz, sc-393084) or normal mouse IgG (Santa Cruz, sc-2025) antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight, followed by washing three times with the PBS and one time with the lysis buffer. All samples were boiled with 2× LDS sample buffer (M00676, GenScript) and then analyzed by Western blotting.
6
Mcl-1 Interaction with BAK in Pancreatic Cancer
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Exponentially growing pancreatic cancer cells were incubated with UMI77 for 48 hours. Cell lysates were prepared with a solution of cold NP-40 lysis buffer (50mM Tris, pH 7.4, 150mM NaCl, 1mM EDTA, 1mM DTT, 0.5% (v/v) NP-40, and Complete protease inhibitor (Roche)). Cellular debris was pelleted at 14,000g for 15 min at 4°C, and the supernatant was then collected and exposed to pre-equilibrated protein A/G Sepharose beads (Santa Cruz Biotechnology). The pre-cleared supernatant was incubated with either Mcl-1 antibody (S-19) or an isotype control (Rabbit IgG; Santa Cruz Biotechnology), overnight at 4°C, followed by the addition of protein A/G Sepharose beads for 2 hours. The beads were pelleted and washed with cold NP-40 lysis buffer followed by elution of the sample from the beads by heating at 95°C for 10 min in SDS loading buffer (60mM Tris pH 6.8, 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol). The immunoprecipitates were subjected to electrophoresis and western analysis using an anti-BAK antibody (CalBiochem).
7
Mass Spectrometry-based Protein Identification
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Protein digestion and MS analysis were executed as previously described47 (link). In brief, proteins were digested with trypsin, vacuum-freeze-dried, and resuspended in anhydrous acetonitrile solution, then desalted with MonoTIPTM C18 Pipette Tip (GL Sciences, Tokyo, Japan). Peptide samples were analyzed with an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Then raw data were analyzed using Proteome Discoverer (Thermo Fisher Scientific) and Spectronaut (Omicsolution Co., Ltd., Shanghai, China) software. Protein and peptide FDRs were set to 1%. For Co-IP, cell lysates were incubated with IgG (Santa Cruz Biotechnology) and protein A/G Sepharose beads (Santa Cruz Biotechnology) at 4 °C for 1 h. The supernatant was mixed with the appropriate primary antibody overnight at 4 °C prior to a 4 h incubation with protein A/G Sepharose beads. After washing with PBS and lysis buffer, the beads were mixed with 5× SDS/PAGE loading buffer for Western blot analysis.
8
Immunoprecipitation of Viral Proteins
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Vero cells seeded in six-well plates were mock-infected or infected with the indicated viruses at an MOI of 5. At 24 h postinfection, the cells were harvested and lysed in NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0) containing protease inhibitors cocktail (Sigma, catalog no. P8340) on ice. After centrifugation at 12,000 rpm for 20 min, the supernatants were collected and precleared with protein A/G Sepharose beads (Santa Cruz Biotechnology, catalog no. sc-2003) and then incubated with 5 μL indicated antibodies and 25 μL protein A&G Sepharose beads overnight at 4°C with gentle rotation. The beads were washed five times with the NP-40 lysis buffer, and the proteins bounded to the beads were separated by SDS-PAGE, followed by Western blotting analysis.
9
Protein Interaction and Ubiquitination Assays
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Cell lysates were incubated with the indicated antibodies at 4 °C overnight and added with 50 μl Protein A/G Sepharose beads (Santa Cruz) for another 4 h. The immunoprecipitates were washed and then processed for western blotting.
For the sequential IP assay, lysates from HEK-293 cells transfected with HA-GSK3β, Myc-Twa1 and Flag-Axin were incubated with anti-Flag antibody bound to protein A/G-agarose beads at 4 °C overnight. The immunoprecipitates were washed and then eluted with Flag peptide for 2 h at 4 °C. The Flag eluates were subsequently incubated with anti-Myc antibody or control IgG at 4 °C overnight and added with protein A/G-agarose beads for another 4 h. The immunoprecipitates were washed and then processed for western blotting.
For the ubiquitination assays, the cells were treated with 25 μM MG132 (Sigma) for 2 h before collection and lysed by RIPA buffer with 10 mM N-ethylmaleimide (Sigma) and a cocktail of protease inhibitors. The prepared lysates were then immunoprecipitated with anti-Flag antibody at 4 °C overnight and subjected to western analysis.
For the sequential IP assay, lysates from HEK-293 cells transfected with HA-GSK3β, Myc-Twa1 and Flag-Axin were incubated with anti-Flag antibody bound to protein A/G-agarose beads at 4 °C overnight. The immunoprecipitates were washed and then eluted with Flag peptide for 2 h at 4 °C. The Flag eluates were subsequently incubated with anti-Myc antibody or control IgG at 4 °C overnight and added with protein A/G-agarose beads for another 4 h. The immunoprecipitates were washed and then processed for western blotting.
For the ubiquitination assays, the cells were treated with 25 μM MG132 (Sigma) for 2 h before collection and lysed by RIPA buffer with 10 mM N-ethylmaleimide (Sigma) and a cocktail of protease inhibitors. The prepared lysates were then immunoprecipitated with anti-Flag antibody at 4 °C overnight and subjected to western analysis.
10
Detecting Nitrosylated Glutamate Transporter
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Samples were incubated with anti-GLT1 antibody overnight and then with protein A/G-Sepharose beads (Santa Cruz Biotechnology). SDS sample buffer (0.05 ml) was added to elute proteins from the protein A/G beads. The eluant was separated on SDS-PAGE (7.5%) and transferred to a nitrocellulose membrane. The membranes were blocked and incubated with anti-nitrotyrosine antibody, further washed and incubated with anti-mouse IgG horseradish peroxidase (1:3,000), and ECL was performed. The membranes were then stripped and reprobed with anti-GLT1 antiserum.
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