Protein g sepharose beads

Manufactured by Santa Cruz Biotechnology

Sourced in United States, China

Protein G Sepharose beads are a type of affinity chromatography resin used for the purification of antibodies. The beads are composed of Sepharose, a polysaccharide matrix, to which the bacterial protein G is covalently coupled. Protein G has a high affinity for the Fc region of immunoglobulins, allowing for the selective capture and purification of antibodies from complex biological samples.

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Lab products found in correlation

1 10 phenanthroline, by Merck Group (3 mentions) Anti ago2 antibody, by Abnova (3 mentions) Laemmli buffer, by Bio-Rad (2 mentions) Tgx precast gel, by Bio-Rad (2 mentions) Geliance 600, by PerkinElmer (2 mentions) Anti flag m2 antibody, by Merck Group (2 mentions) Horseradish peroxidase conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions) Gradient sds polyacrylamide gel, by Bio-Rad (1 mentions) Errα antibody, by Cell Signaling Technology (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Clarity western ecl substrate, by Bio-Rad (1 mentions) Ezview red anti flag m2 affinity gel, by Merck Group (1 mentions) Superfect reagent, by Qiagen (1 mentions) Anti c ebpβ c 19 antibody sc 150, by Santa Cruz Biotechnology (1 mentions) Fluorescein isothiocyanate dextran, by Merck Group (1 mentions) Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions) Anti p p38, by Cell Signaling Technology (1 mentions) Anti erk, by Cell Signaling Technology (1 mentions) Anti β actin antibody, by Merck Group (1 mentions)

Close spelling variants of this product

Protein A/G-Sepharose bead suspension Protein G Sepharose Protein G beads Sepharose beads

25 protocols using protein g sepharose beads

Quantifying Ago2-bound Snail1 mRNA in HK2 cells

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Twenty‐four hours after transfection with miR‐30c mimics or miR‐con, HG‐treated HK2 cells were lysed and then immunoprecipitated with anti‐Ago2 antibody or IgG (Santa Cruz Biotech) using protein G Sepharose beads (Santa Cruz Biotech), as described previously (Beitzinger & Meister, 2011; Li et al., 2016; Yin et al., 2016). After washing, a small aliquot of beads was transferred to a new tube for Western blot using anti‐Ago2 antibody to confirm efficient precipitation of Ago protein complexes. The remaining products were extracted with TRIzol, and the levels of Snail1 mRNA were quantified by real‐time PCR. Lysates of renal cortex of db/db mice with different rAAVs treatments were also analyzed.

Zhao Y., Yin Z., Li H., Fan J., Yang S., Chen C, & Wang D.W. (2017). MiR‐30c protects diabetic nephropathy by suppressing epithelial‐to‐mesenchymal transition in db/db mice. Aging Cell, 16(2), 387-400.

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Immunoprecipitation and Western Blotting of PGC1α

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Cell extracts were prepared using Triton lysis buffer and incubated (16 hrs., 4°C) with either 3 μg non-immune control rabbit IgG (Cell Signaling #2729) or with ERRα antibody (Cell Signaling #13826) to 500 uL of cell lysate. Immunocomplexes were isolated using Protein G Sepharose beads (Santa Cruz #SC2002, Santa Cruz, CA) and washed 4–5 times with lysis buffer. Bead pellets were resuspended and boiled in β-mercaptoethanol containing Laemmli sample buffer, separated in 4% to 12% gradient SDS-polyacrylamide gels (Bio-Rad #456–8094), transferred to nitrocellulose membranes (Bio-Rad #170–4271, Hercules, CA) and incubated with rabbit anti PGC1α antibody with 1:1000 dilution. Immunocomplexes were visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling, #7074) and detected with a Clarity western ECL substrate (Bio-Rad #170–5061).

Kant S., Tran K.V., Kvandova M., Caliz A.D., Yoo H.J., Learnard H., Dolan A.C., Craige S.M., Hall J.D., Jiménez J.M., St. Hilaire C., Schulz E., Kröller-Schön S, & Keaney JF J.r. (2021). PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1. Arteriosclerosis, thrombosis, and vascular biology, 42(1), 19-34.

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Enrichment and Immunoprecipitation of PSMA1

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Cells were lysed with 25 mM HEPES, pH 7.4, 10% glycerol, 5 mM MgCl2, 1 mM ATP, and 1:400 protease-inhibitor mixture (Calbiochem), then homogenized through freeze–thaw cycles and passed through a needle. The lysates were cleared by 30-min centrifugation at 21,130g at 4 °C. Pellets were lysed again with 0.5 mM ammonium persulfate to enrich nuclear fraction followed be centrifugation. Mixed lysates were treated with 2 mM 1,10-phenanthroline (Sigma), cross-linked with 0.5 mM DSP (Thermo Fisher Scientific) for 30 min at room temperature, and quenched in 100 mM Tris-HCl, pH 8, 5 mM L-cysteine for 10 min at room temperature. For immunoprecipitation, the lysates were then incubated with Protein G–Sepharose beads (Santa Cruz) with antibodies to PSMA1 and eluted with 100 mM Tris-HCl, pH 8, 8M urea and 50 mM DTT for 30 min at 37 °C. Subsequently, 1% trifluoroacetic acid (TFA) was added. Aliquots of each elution fraction were analyzed by SDS–PAGE to evaluate yield and purity.

Benyair R., Giridharan S.S., Rivero-Ríos P., Hasegawa J., Bristow E., Eskelinen E.L., Shmueli M.D., Fishbain-Yoskovitz V., Merbl Y., Sharkey L.M., Paulson H.L., Hanson P.I., Patnaik S., Al-Ramahi I., Botas J., Marugan J, & Weisman L.S. (2023). Upregulation of the ESCRT pathway and multivesicular bodies accelerates degradation of proteins associated with neurodegeneration. Autophagy reports, 2(1), 2166722.

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MIZ1 Interactome Analysis in HEK293T

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HEK293T cells were transfected with pcDNA-HA-HA-MYC, pcDNA-HA-MYCN or pcDNA-HA-HA-MYCL in combination with pcDNA-MIZ1. Two days post-transfection, cells were harvested and subjected to MIZ1 IP using anti-MIZ1 antibody (sc-139685, Santa Cruz Biotechnology, 4 µg). Antibody–protein complexes were captured using 20 µl protein G sepharose beads (Santa Cruz Biotechnology). Immunoprecipitates were then analyzed by western blot.

Dammert M.A., Brägelmann J., Olsen R.R., Böhm S., Monhasery N., Whitney C.P., Chalishazar M.D., Tumbrink H.L., Guthrie M.R., Klein S., Ireland A.S., Ryan J., Schmitt A., Marx A., Ozretić L., Castiglione R., Lorenz C., Jachimowicz R.D., Wolf E., Thomas R.K., Poirier J.T., Büttner R., Sen T., Byers L.A., Reinhardt H.C., Letai A., Oliver T.G, & Sos M.L. (2019). MYC paralog-dependent apoptotic priming orchestrates a spectrum of vulnerabilities in small cell lung cancer. Nature Communications, 10, 3485.

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Immunoprecipitation for Protein Complex Analysis

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Immunoprecipitations were performed by incubating the same amount of the relevant whole-cell lysates with the appropriate antibodies (500–1000 μg of whole cell lysate, 2 μg of antibody) overnight at 4 °C with gentle agitation to allow immunocomplexes to form. Prior to incubation with the specified antibodies, samples were precleared by incubation with protein G-Sepharose beads (sc-2002, Santa Cruz) and 2 μg of α-mouse IgG (I5381, Sigma), for 1 h at 4 °C. Following the overnight incubation and when applicable, the immunocomplexes were collected by the addition of protein G-Sepharose beads for 3 h at 4 °C and washed six times with lysis buffer. Proteins were eluted by boiling in 2× SDS loading buffer and eluates were resolved on a 10% SDS-PAGE for immunoblotting with relevant antibodies. Unless otherwise indicated, FLAG-tagged proteins were immunoprecipitated with EZview Red ANTI-FLAG M2 Affinity Gel (F2426, Sigma). Otherwise, the following antibodies were used: monoclonal mouse α-PAK4 (clone OTI1C7 #CF807297, Origene), monoclonal mouse α-RELB (clone 17.3 #LS-C354950–100, LSBio), and α-mouse IgG (I5381, Sigma) as control.

Costa T.D., Zhuang T., Lorent J., Turco E., Olofsson H., Masia-Balague M., Zhao M., Rabieifar P., Robertson N., Kuiper R., Sjölund J., Spiess M., Hernández-Varas P., Rabenhorst U., Roswall P., Ma R., Gong X., Hartman J., Pietras K., Adams P.D., Defilippi P, & Strömblad S. (2019). PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer. Nature Communications, 10, 3589.

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Investigating C/EBPβ Protein Interactions

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MC3T3 cells were transfected with the 3xFlag-C/EBPβ-LAP*-coding vector or its mutant variant 3xFlag-C/EBPβ-LAP*R3L using Superfect reagent (Qiagen, Germany) and nuclear extracts prepared as described previously (Paredes et al., 2004 (link)). Coimmunoprecipitations were reported before (Sierra et al., 2003 (link)). Flag-tagged C/EBPβ-LAP* or C/EBPβ-LAP*R3L proteins were immunoprecipitated using anti-Flag M2 antibody (F3165, Sigma Aldrich). The immunocomplexes were captured using Protein G-Sepharose beads (Santa Cruz Biotechnology). The immunoprecipitated proteins were detected by Western blot using the following antibodies: anti-Brg1 serum (donated by Dr. Anthony Imbalzano), anti-Ini1 H-300 antibody (sc-13055, Santa Cruz Biotechnology), anti-C/EBPβ C-19 antibody (sc-150, Santa Cruz Biotechnology), and anti-Flag M2 antibody (F3165, Sigma Aldrich).

Aguilar R., Grandy R., Meza D., Sepulveda H., Pihan P., van Wijnen A.J., Lian J.B., Stein G.S., Stein J.L, & Montecino M. (2014). A Functional N-terminal Domain in C/EBPβ-LAP* is Required for Interacting with SWI/SNF and to Repress Ric-8B Gene Transcription in Osteoblasts. Journal of cellular physiology, 229(10), 1521-1528.

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Antibodies and Reagents for Cell Signaling

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The following antibodies were used in this study: anti-CD146 rabbit polyclonal antibody and anti-CD146 mouse mAb of AA1 were generated in our lab. Anti-β-actin antibody, HRP-conjugated goat anti-mouse and anti-rabbit IgG antibodies were from Sigma-Aldrich. Anti-human IgG Fc and anti-His antibodies were from ZSGB-BIO. Anti-VEGFR-3 antibody and anti-VEGF-C antibody were from Abcam. Anti-AKT, anti-p-AKT, anti-p38, anti-p-p38, anti-ERK and anti-p-ERK antibodies were from Cell Signaling Technology. Goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 555 were from Invitrogen.
The following reagents were used: recombinant human VEGFR-3-Fc, human VEGF-C, Fc-CD146 and sCD146 were from Sino Biological. Human VEGF-C156S was from R&D. ERK1/2 inhibitor, SCH772984, and p38 inhibitor, FHPI, were from Selleckchem. Growth factor-reduced Matrigel was from BD Biosciences. Fugene HD, DAPI and protease inhibitor cocktails were from Roche. Protein G sepharose beads was from Santa Cruz. Enhanced Chemiluminescence Assay Kit for WB was from Pierce. Cell Counting Kit-8 (CCK-8) for cell proliferation assay was from Dojindo. Human Fc, Carboxymethylcellulose for spheroid assay and Fluorescein isothiocyanate-dextran average molecular weight 2000 KD and Tetramethylrhodamine-dextran average molecular weight 70 KD were from Sigma-Aldrich.

Yan H., Zhang C., Wang Z., Tu T., Duan H., Luo Y., Feng J., Liu F, & Yan X. (2017). CD146 is required for VEGF-C-induced lymphatic sprouting during lymphangiogenesis. Scientific Reports, 7, 7442.

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Quantification of Immune Sensor Proteins by Western Blot

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RIG-I, TLR3, and TLR7 protein expression were quantified by Western blot assay. Whole-cell lysates were treated with radioimmunoprecipitation assay buffer containing proteinase inhibitor cocktail (Sigma-Aldrich). Total proteins were biotinylated with 0.2 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL, USA), immunoprecipitated for 90 min at 4°C with specific monoclonal antibodies (anti-RIG-I, Clone OTI6C1; anti-TLR3, Clone 27N3D4; anti-TLR7, Clone NBP2-24905; and anti-β actin, Clone NB600-501) (Novus Biologics, Centennial, CO, USA), and incubated overnight at 4°C with protein-G Sepharose beads (Santa Cruz Biotechnology, Dallas, TX, USA). The samples were resuspended in 20 µl Laemmli buffer (Bio-Rad). Twenty micrograms of immunoprecipitated proteins were loaded in each well and evaluated in denaturating conditions in 10 per cent TGX precast gel (Bio-Rad), with subsequent electroblotting transfer onto a Polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). The membrane was incubated with horseradish peroxidase-conjugated streptavidin (Amersham Biosciences, Piscataway, NJ, USA) and developed with the enhanced chemiluminescence kit (Amersham Biosciences). The images were acquired by Geliance 600 (Perkin Elmer, Waltham, MA, USA).

Caccuri F., Messali S., Bortolotti D., Di Silvestre D., De Palma A., Cattaneo C., Bertelli A., Zani A., Milanesi M., Giovanetti M., Campisi G., Gentili V., Bugatti A., Filippini F., Scaltriti E., Pongolini S., Tucci A., Fiorentini S., d’Ursi P., Ciccozzi M., Mauri P., Rizzo R, & Caruso A. (2022). Competition for dominance within replicating quasispecies during prolonged SARS-CoV-2 infection in an immunocompromised host. Virus Evolution, 8(1), veac042.

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Immunoprecipitation and RNA Extraction

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Lysed cell extracts were immunoprecipitated with anti-Ago2 antibody (Abnova, Taiwan, China) or immunoglobulin G (IgG) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using protein G Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as described.¹⁰ (link) After eluted from the beads, bound RNA were extracted with TRIzol and quantified by quantitative real-time PCR.

Li H., Dai B., Fan J., Chen C., Nie X., Yin Z., Zhao Y., Zhang X, & Wang D.W. (2019). The Different Roles of miRNA-92a-2-5p and let-7b-5p in Mitochondrial Translation in db/db Mice. Molecular Therapy. Nucleic Acids, 17, 424-435.

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CD146 and Netrin-1 Interaction Assay

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HEK293 cells were co-transfected with plasmids encoding the full-length CD146, netrin-1 or the truncation mutants using Fugene HD (Roche). 48 h post transfection, the cells were lysed in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 0.1% SDS, 0.5% deoxycholate, 0.1% NP-40, 1 mM PMSF, protease inhibitor cocktails). Then the cell lysates were incubated with the control mIgG, anti-CD146 mAb AA1 or anti-netrin-1 mAb for 4 h at 4 °C, and immunoprecipitation was carried out using protein G sepharose beads (Santa Cruz). The cell lysates and precipitates were analyzed by western blotting using chemiluminescence imaging system (ChemiScope 3400; Clinx, China).

Tu T., Zhang C., Yan H., Luo Y., Kong R., Wen P., Ye Z., Chen J., Feng J., Liu F., Wu J.Y, & Yan X. (2015). CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development. Cell Research, 25(3), 275-287.

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