Approximately 107 HFFs were lysed with RIPA buffer and total lysate was precleared with 50 μL protein A/G agarose bead slurry (Santa Cruz) for 1 h. The precleared lysates were quantified as described above and 500 μg to 1 mg of total lysate was incubated overnight with anti-ubiquitin (1 μg), anti-TNKS (2 μg), or anti-Axin1 (2 μg) antibody. The antibody–lysate complexes were incubated with protein A/G beads (Santa Cruz) at 4 °C and washed three times with RIPA buffer. Samples were boiled in SDS sample buffer, and the supernatant was analyzed on SDS-PAGE gel after immunoblotting as described previously. Then, 1%–5% of the cell lysate used for IP was loaded on gels as “Input”.
Protein a g agarose bead slurry
Manufactured by Santa Cruz Biotechnology
Protein A/G agarose bead slurry is a versatile affinity resin designed for the purification of immunoglobulins and other proteins. The resin consists of Protein A and Protein G covalently coupled to agarose beads, which can bind to the Fc region of immunoglobulins, allowing for efficient capture and isolation of these proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g bead, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Anti ar, by Santa Cruz Biotechnology (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Anti fkbp52, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Illustrator cc 2018, by Adobe (1 mentions)
Image studio software, by LI COR (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
5 protocols using protein a g agarose bead slurry
1
Ubiquitin, TNKS, and Axin1 Co-Immunoprecipitation
2
Protein A/G Pulldown of Viral DNA
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Two hundred microliters of serum sample was first mixed with 300 μl of TNE buffer, and then 15 μl of protein A/G agarose bead slurry (Santa Cruz) was added to the mixture, followed by incubation overnight at 4°C in a sample mixer. Subsequently, protein A/G agarose beads were washed three times with TNE buffer, and viral DNA in input serum samples (40 μl) and agarose bead pulldown mixtures were extracted and subjected to Southern blot analysis.
3
Investigating AR-β-catenin Interactions
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Co-immunoprecipitations were performed from extracts of LNCaP cells that were transfected with FKBP52 siRNA or 293T cells that were co-transfected with Gal4-AR LBD and β-catenin expression plasmids. Transfected or co-transfected cells maintained in 10% Charcoal-stripped serum were treated with and without 10 nM DHT and 30μ M MJC13 following transfection, and harvested in RIPA cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.5 mM EGTA, 1% NP-40, Protease inhibitor cocktail (Roche)). Whole-cell lysate was incubated with 2 μ g of anti-β-catenin or anti-Gal4 antibody (Santa Cruz Biotechnology) for 4 hours at 4ºC, and was further incubated for another 12 hours after the addition of 30 μ l of protein A/G agarose bead slurry (Santa Cruz Biotechnology). Agarose beads were washed three times with RIPA buffer at 4ºC, and bound proteins were separated by SDS-PAGE. Proteins on the gels were transferred to a PVDF membrane, subjected to Western blot analysis with anti-AR (Santa Cruz Biotechnology), anti-FKBP52 (Cell Signaling Technologies), anti-β-catenin (Santa Cruz Biotechnology), and anti-GAPDH (Cell Signaling Technologies) antibodies, and then detected with an ECL kit (Amersham Pharmacia).
4
Nuclear Protein Immunoprecipitation Workflow
5
Protein Immunoprecipitation and Western Blotting
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Cells were lysed in high-salt buffer (300 nM NaCl, 50 mM Tris-HCl [pH 8], 10% glycerol, and 0.2% NP-40) supplemented with protease inhibitors (Sigma-Aldrich #04693132001) and sonicated 5 min at 4 °C with a Bioruptor in 30” ON-OFF cycles. After centrifugation at 16,000×g for 15 min, soluble material was quantified by Bradford assay (Bio-rad #5000006), and 1 mg of protein was used for each immunoprecipitation (IP), or 30–50 μg of protein was loaded onto SDS-PAGE gels for western blotting. IP samples were incubated overnight with 5 μg of antibody (see Supplementary Data 6 for a list of antibodies used) followed by 30 μl of protein A/G agarose bead slurry (Santa Cruz #sc-2003) for 2 h. IP material was washed 3× with high-salt buffer and eluted with Laemmli buffer, then loaded for SDS-PAGE. Western blotting was performed using standard protocols and imaged on an Odyssey CLx imaging system (Li-COR), and various exposures within the linear range captured using ImageStudio software (Li-COR). Images were rotated, resized, and cropped using Adobe Photoshop CC 2018 and imported into Adobe Illustrator CC 2018 to be assembled into figures. Unprocessed images for all western blots in main figures are provided in Supplementary Fig. 10 .
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