Protein a g plus sepharose beads

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Protein A/G Plus-Sepharose beads are a solid-phase affinity matrix designed for the purification of antibodies. They contain a covalent coupling of recombinant Protein A and Protein G to Sepharose beads, providing a high-capacity platform for the capture of immunoglobulins from various sources.

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Lab products found in correlation

Image studio lite, by LI COR (1 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Complete mini edta free and phosstop, by Roche (1 mentions) Quick western kit, by LI COR (1 mentions) Gradient gel, by Thermo Fisher Scientific (1 mentions) Odyssey infrared scanner, by LI COR (1 mentions) Image studio software, by LI COR (1 mentions)

3 protocols using protein a g plus sepharose beads

STAT3 and DEC1 Interaction Analysis

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DLD-1 cells were seeded in 60 mm dishes for overnight and then treated with IL-6 (50 ng/ml) or the same volume of PBS for 24 h. Cell lysates were prepared in ice-cold RIPA buffer (1 ml). Cell lysates (500 μg) were incubated with anti-STAT3 or anti-DEC1 antibody (1 μl) at 4 °C for overnight. The immune complexes were precipitated with 20 μl of Protein A/G Plus-Sepharose beads (Santa Cruz, Dallas, USA, Cat: sc-2003) at 4 °C for 4 h. The beads were washed for 8 times with ice-cold buffer (50 mM Tris/HCl, pH 7.4 and 150 mM NaCl). The immuno-precipitates were analyzed for the presence of DEC1 or STAT3 by Western blotting with anti-DEC1 and anti-STAT3 antibodies. The cell lysates (5 μg), prior to immunoprecipitation, were also analyzed for the levels of DEC1 and STAT3.

Shan E., Hao Y., Wang H., Zhang Z., Hu J., Wang G., Liu W., Yan B., Hiroaki H, & Yang J. (2022). Differentiated embryonic chondrocyte expressed gene-1 (DEC1) enhances the development of colorectal cancer with an involvement of the STAT3 signaling. Neoplasia (New York, N.Y.), 27, 100783.

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Detecting NLRP3 Inflammasome Activation

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BMDM cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in modified lysis buffer (50 mM Tris-Cl (pH 7.4), 1% Nonidet P-40, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL each of aprotinin and leupeptin, and 1 mM Na₃VO₄). Protein (1 mg) of cell extracts was incubated with 2 μg of anti-NLRP3 antibodies for 2 h at 4 °C, followed by incubation with 40 μL of Protein A/G Plus-Sepharose beads (Santa Cruz Biotechnology, Inc.) overnight at 4 °C. The beads were washed with lysis buffer, and immune complexes were eluted by boiling in 2× sodium dodecyl sulfate (SDS) Laemmli loading buffer for 5 min prior to immunoblotting analyses with indicated antibodies. For immunoblotting, the immune complexes were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were immunoblotted with antibodies for NLRP3 or ASC, followed by HRP-conjugated secondary antibodies. Specific bands were visualized using Pierce ECL Western Blotting Substrate from ThermoFisher Scientific (Waltham, MA). Band intensities of immunoprecipitated ASC or NLRP3 panel were quantified using Image Studio LITE (LI-COR biotechnology) software. The ratios of ASC/NLRP3 association were calculated with PBS control group set as 1.

Guo C., Fulp J.W., Jiang Y., Li X., Chojnacki J.E., Wu J., Wang X.Y, & Zhang S. (2017). Development and Characterization of a Hydroxyl-Sulfonamide Analogue, 5-Chloro-N-[2-(4-hydroxysulfamoyl-phenyl)-ethyl]-2-methoxy-benzamide, as a Novel NLRP3 Inflammasome Inhibitor for Potential Treatment of Multiple Sclerosis. ACS chemical neuroscience, 8(10), 2194-2201.

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Immunoprecipitation and Western Blotting

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Cells were collected in lysis buffer (1 mM EGTA, 1 mM EDTA, 10 mM Tris [pH 7.4], 150 mM sodium chloride, 1% deoxycholate, 1% NP-40, and supplemented with protease and phosphatase inhibitor cocktail tablets [cOmplete mini EDTA-free and PhosStop; Roche]). Lysates were precleared with 10 μl of protein A/G PLUS Sepharose Beads (Santa Cruz Biotechnology) for 1 h at 4°C. The precleared lysates were then incubated with 20 μl of protein A/G PLUS Sepharose Beads, which had previously been coupled with the appropriate primary antibody for 2 h at 4°C on an orbital shaker. The beads were washed three times with lysis buffer supplemented with protease and phosphatase inhibitors. The immunoprecipitation complexes were eluted from the beads by boiling in 2× SDS buffer for 5 min.
For Western blotting analyses, protein lysates/coimmunoprecipitation complexes were resolved on a 4%–12% gradient gel (Invitrogen) and blotted on a nitrocellulose membrane with the appropriate primary antibodies (1:1,000) and IRDye-conjugated anti-mouse, anti-goat, or anti-rabbit secondary antibodies or the Quick Western Kit (926-69100; LI-COR), as appropriate. Images were acquired on a LI-COR Odyssey infrared scanner. Densitometric quantification of bands was performed using Image Studio software (LI-COR).

Keen A.N., Payne L.A., Mehta V., Rice A., Simpson L.J., Pang K.L., del Rio Hernandez A., Reader J.S, & Tzima E. (2022). Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells. The Journal of Cell Biology, 221(2), e202005213.

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