Protein a g agarose

Manufactured by Abmart

Sourced in China

Protein A/G agarose is a chromatographic resin designed for the purification of antibodies. It contains a mixture of Protein A and Protein G, which are bacterial surface proteins that bind to the Fc region of immunoglobulins. This resin can be used to capture and isolate antibodies from complex biological samples.

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Lab products found in correlation

Anti flag m2 affinity gel a2220, by Merck Group (1 mentions) Anti myc m20002, by Abmart (1 mentions) Uric acid u2625, by Merck Group (1 mentions) Anti ezrin ab4069, by Abcam (1 mentions) Anti flag m20008, by Abmart (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Di 4 aneppdhq, by Thermo Fisher Scientific (1 mentions) Murine il 4, by Thermo Fisher Scientific (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions) β mercaptoethanol, by Merck Group (1 mentions) Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions) Akt antibody, by Cell Signaling Technology (1 mentions) P38 mapk antibody, by Cell Signaling Technology (1 mentions) β catenin antibody, by Cell Signaling Technology (1 mentions) Il 33, by R&D Systems (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Inos antibody, by Abcam (1 mentions) F4 80 antibody, by Santa Cruz Biotechnology (1 mentions) Il 33 antibody, by R&D Systems (1 mentions)

Spelling variants of this product

Protein A/G agarose beads (12 citations) Protein A/G (2 citations) Protein A/G PLUS-agarose beads (2 citations)

10 protocols using protein a g agarose

Immunoprecipitation and Ubiquitination Assay Protocol

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Harvested cells were lysed with a lysis buffer (20 mM HEPES pH 8.0, 0.2 mM EDTA, 5% glycerol, 150 mM NaCl, and 1% NP-40) on ice for 15 min. The lysate was then sonicated on ice at 10% power for 2 min. After centrifuging at 13 500 rpm for 15 min at 4°C, the supernatants were precleared by incubation with 20 μl protein A/G agarose (Abmart, #A10001M) and IgG (Santa Cruz, #sc-2025) for 1 h at 4°C. The samples were then centrifuged at 1000 rpm for 3 min at 4°C. The supernatants were incubated with antibodies overnight. Then, protein A/G agarose was added to the supernatants and incubated for 2 h at 4°C. The precipitates were washed three times with the lysis buffer, boiled for 10 min with 2× SDS sample buffer and resolved by SDS-PAGE.
For the precipitation of ubiquitinated DDB2-FLAG under denaturing conditions, cells were lysed in SDS lysis buffer (50 mM Tris–HCl pH 6.8, 5 mM DTT and 1% (w/v) SDS), followed by sonication on ice at 10% power for 4 min. After centrifuging at 13 500 rpm for 15 min at 4°C, the supernatant was washed four times with NP-40 buffer (25 mM Tris–HCl pH 8.0, 0.3 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) NP-40 and EDTA-free protease inhibitor cocktail) and incubated with 20 μl protein A/G agarose (Abmart, #A10001M) or IgG (Santa Cruz, #sc-2025) for 1 h at 4°C, followed by performance of the procedures described above.

Geng A., Tang H., Huang J., Qian Z., Qin N., Yao Y., Xu Z., Chen H., Lan L., Xie H., Zhang J., Jiang Y, & Mao Z. (2020). The deacetylase SIRT6 promotes the repair of UV-induced DNA damage by targeting DDB2. Nucleic Acids Research, 48(16), 9181-9194.

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Ago2 Immunoprecipitation and miRNA Analysis

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Human anti-Ago2 antibody (Abnova) was first bound to protein A/G-Agarose (Abmart) in PBS for 30 min at 4°C. Treated F81 cells were harvested, washed and solubilized in RIPA lysis buffer (HX-BIO) and PSMF for 30 min on ice, then centrifuged at 12,000 g for 30 min to clarify the supernatant. The latter was then added to the Ago2/Agarose conjugate and incubated for 4 h at 4°C. Incubation of the supernatant with normal mouse IgG (MBL) was used as a negative control. RNA bound to the Ago2 protein was dissociated with Trizol reagent and reverse transcribed. TfR, miR-320a and miR-140 were quantified by qPCR analysis, with β-actin and U6 small RNA as internal controls.

Sun J.Z., Wang J., Wang S., Yuan D., Li Z., Yi B., Hou Q., Mao Y, & Liu W. (2014). MicroRNA miR-320a and miR-140 inhibit mink enteritis virus infection by repression of its receptor, feline transferrin receptor. Virology Journal, 11, 210.

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Signaling pathway analysis in T cells

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Anti-Moesin (ab151542) (1:1000), anti-Ezrin (ab4069) (1:200), anti-Radixin (ab52495) (1:1000), anti-phospho-ERM (ab76247) (1:1000), and anti-CD8α (ab209775) were purchased from Abcam. anti-Zap-70 (2705) (1:1000), anti-Syk (2712) (1:1000), anti-phospho-Erk1/2 (8544, 9101) (1:1000), anti-Erk1/2 (9102) (1:1000), anti-phospho-Akt (9271) (1:800), anti-Akt (9272) (1:1000), anti-phospho-P38 (9211) (1:1000), anti-P38 (9212) (1:1000), anti-rabbit-IgG (7074), and anti-mouse-IgG (7076) were purchased from CST. Anti-phospho-tyrosine (05–321) (1:1000) was purchased from Merck. Anti-GAPDH (BE0023) (1:3000) was purchased from EASYBIO. Anti-Myc (M20002) (1:1000), anti-Flag (M20008) (1:1000), and Protein A/G-Agarose (A10001) were purchased from Abmart. Anti-Flag M2 affinity gel (A2220) and uric acid (U2625) were purchased from Sigma-Aldrich. Polystyrene microspheres (19822) were purchased from Polysciences. Piceatannol (527948), Syk Inhibitor (574711), Syk Inhibitor IV, BAY 61-3606 HCL (574714), R406 (5.05819.0001), Wortmannin (681676), and LY294002 (440204) were purchased from Calbiochem. AZD0530 (S1006) and PP2 (S7008) were purchased from Selleck. Bodipy FL-PI(4,5)P2 (C-45F6) were purchased from Echelon Biosciences. TopFluor TMR-PC (810180C) and TopFluor TMR-PE (810241C) were purchased from Avanti Lipids. Geneticin (10131027) was purchased from ThermoFisher.

Mu L., Tu Z., Miao L., Ruan H., Kang N., Hei Y., Chen J., Wei W., Gong F., Wang B., Du Y., Ma G., Amerein M.W., Xia T, & Shi Y. (2018). A phosphatidylinositol 4,5-bisphosphate redistribution-based sensing mechanism initiates a phagocytosis programing. Nature Communications, 9, 4259.

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Immunoprecipitation and SDS-PAGE Analysis

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The cells were also lysed in RIPA buffer-containing PMSF(1*10⁶ cell/150 μL). The homogenized samples were centrifuged to collect the supernatants. The cell lysate was cleared by adding 20 μL of Protein A/G-Agarose (Abmart, China, Shanghai) slurry (50%) per 1 mL of cell lysate and incubating at 4°C for 10 min on a rotator. The Protein A/G-Agarose was removed by centrifugation at 14,000 g at 4°C for 5 min. The supernatant was transferred to a fresh centrifuge tube. The 5 μL BIP or COX-2 antibody of cell lysate was added. The cell lysate/antibody mixture was gently rotated for either 2 h or overnight at 4°C on a rotator. The immunocomplex was captured by adding 30–40 μL Protein A/G-Agarose slurry (15–20 μL packed beads) and gently rotated on a rotator for 1 or 2 h at 4°C. The agarose beads were collected by centrifuging for 3 min at 1000 rpm. The supernatant was discarded and the beads were washed three times with an 800 μL ice-cold RIPA buffer. The agarose beads were resuspended in 30–60 μL 1× SDS loading buffer and mixed gently. The agarose beads were boiled for 10 min at 100°C to dissociate the immunocomplexes from the beads. The beads were collected by centrifugation and SDS-PAGE was performed with the supernatant. Unused samples may be stored at −20°C for later use.

Wang H., Yao J., Chen Y., Wang Y., Liu Y., Liao Y., Liang Z., Dong Y.H., Qu M., Ge X, & Zhou X. (2022). Gut dysbacteriosis attenuates resistance to Mycobacterium bovis infection by decreasing cyclooxygenase 2 to inhibit endoplasmic reticulum stress. Emerging Microbes & Infections, 11(1), 1806-1818.

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Murine Cytokine and Lipid Protocol

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Murine GM-CSF (315-03-1000) and murine IL-4 (214-14-000) were obtained from Peprotech; protein A/G agarose was obtained from Abmart; sodium orthovanada was obtained from Yeason Biotech; phorbol 12-myristate 13-acetate (PMA)/12-O-tetradecanoylphorbol 13-acetate (TPA) mixture solution (S1819) and PMSF were obtained from Beyotime biotech; Lipofectamine 2000 (11668019) was obtained from Thermo Fisher Scientific; β-mercaptoethanol (07604) and DMSO (V900090) were obtained from Sigma-Aldrich. Biotin Labeling Kit (E-LK-B002) was obtained from Elabscience. Di-4-ANEPPDHQ (D36802) was from Thermo Fisher Scientific. Total cholesterol (TC) content assay kit (BC1985) was obtained from Solarbio. Alexa Flour 647-conjugated Annexin-5/PI kit was purchased from 4A biotech.

Xu Y., Song D., Wang W., Li S., Yue T., Xia T, & Shi Y. (2023). Clec12a inhibits MSU-induced immune activation through lipid raft expulsion. Life Science Alliance, 6(9), e202301938.

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Isolation and Analysis of IL-33 in Murine Skin Wounds

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2 mm mouse skin taken from the margin of murine wounds or cells stimulated as described was lysed by using RIPA buffer (pH 7.4) containing protease inhibitor cocktail (Roche). To concentrate IL-33 from cell culture medium, mouse IL-33 antibody (R&D) immunocomplexed to Protein A/G agarose (Abmart) was used to capture IL-33. 10 µg of total protein was used for Western blot. IL-33, iNOS, p38 MAPK, AKT and β-catenin were detected by immunoblot with IL-33 antibody (R&D), iNOS antibody (Abcam), p38 MAPK antibody (Cell Signaling), AKT antibody (Cell Signaling) and β-catenin antibody (Cell signaling), respectively.
5 µm of formalin-fixed, paraffin-embedded tissue sections was mounted on glass slides and used for immunohistochemistry and staining. 4% PFA was used to fixate the samples. After 10-min fixation and subsequent pretreated with antigen retrieval solution, the sections were stained with IL-33 (R&D) antibody or iNOS (Abcam) antibody and F4/80 antibody (Santa Cruz) or Ly6G/Gr antibody (Abcam). The sections were reprobed with anti-Goat IgG FITC (Jackson immunoresearch) or anti-Rat IgG TRITC conjugate antibody (KPL), and then mounted in ProLong Gold antifade reagent with DAPI (Invitrogen) and visualized them by the microscope (Leica).

Li C., Li H., Jiang Z., Zhang T., Wang Y., Li Z., Wu Y., Ji S., Xiao S., Ryffel B., Radek K.A., Xia Z, & Lai Y. (2014). Interleukin-33 Increases Antibacterial Defense by Activation of Inducible Nitric Oxide Synthase in Skin. PLoS Pathogens, 10(2), e1003918.

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Immunoprecipitation and Cell Lysis Protocol

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Cells were harvested, washed once with 1 × PBS, and lysed with lysis buffer (20 mM HEPES, pH 8.0; 0.2 mM EDTA; 5% glycerol; 150 mM NaCl; 1% NP-40) for 15 min on ice. The lysate was then sonicated on ice for 3 min before being centrifuged at 13 500 rpm for 15 min at 4°C. The supernatant was then prepared for immunoprecipitation by two methods. In the first method, the supernatant was pretreated with protein A/G agarose (Abmart, Cat. # A10001M) and IgG (Santa Cruz, #sc-2025) for 1 h at 4°C. After centrifugation, the supernatant was collected and incubated with the indicated antibodies overnight. Afterward, the samples were incubated for 2 h at 4°C after 30 μl of protein A/G agarose was added to the mixture. In the other method, cell lysates were incubated with anti-DYKDDDK magnetic beads (Selleck, Cat. # B26102) or GFP-Trap (ChromoTek, Cat. # gta-100) overnight at 4°C. All precipitated samples were washed three times with lysis buffer and boiled for 10 min with 2 × sample buffer (1% SDS; 10 mM EDTA; 50 mM Tris–HCl, pH 8.1; and protease inhibitors). Whole-cell lysates were used as input as a control.

Geng A., Sun J., Tang H., Yu Y., Wang X., Zhang J., Wang X., Sun X., Zhou X., Gao N., Tan R., Xu Z., Jiang Y, & Mao Z. (2024). SIRT2 promotes base excision repair by transcriptionally activating OGG1 in an ATM/ATR-dependent manner. Nucleic Acids Research, 52(9), 5107-5120.

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Identifying TRIM25 Viral Protein Interactions

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DF-1 cells were seeded in 6-well plates and transfected with the indicated plasmids. Then, the cells were washed thrice with ice-cold PBS and lysed in 500 μL Western blotting and IP lysis buffers (P0013, Beyotime, China) for 30 min. After 12,000×g centrifugation, the supernatants of cell lysates were incubated with 1 μg anti-Flag mouse monoclonal antibody (mAb) or control mouse IgG for 6–8h or overnight. Afterward, 40 μL protein A/G agarose (A10001, Abmart) was added to the lysate mixture for 6–8h. The beads were collected by centrifugation at 3,000 ×g for 5 min at 4 °C and washed five times with ice-cold PBS. To identify the viral proteins related to TRIM25, 2 μg pHA-VP1, pHA-VP2, pHA-VP3, pHA-VP4, or pHA-VP5 and 2 μg pFlag-TRIM25 were co-transfected, respectively, and the search was then conducted following the above instruction. Furthermore, the relationship between TRIM25 and VP3 was detected in both directions by co-transfecting pFlag-plasmids and pMyc-plasmids.

Wang S., Yu M., Liu A., Bao Y., Qi X., Gao L., Chen Y., Liu P., Wang Y., Xing L., Meng L., Zhang Y., Fan L., Li X., Pan Q., Zhang Y., Cui H., Li K., Liu C., He X., Gao Y, & Wang X. (2021). TRIM25 inhibits infectious bursal disease virus replication by targeting VP3 for ubiquitination and degradation. PLoS Pathogens, 17(9), e1009900.

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NLRP3 Inflammasome Activation and Immunoprecipitation

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For endogenous immunoprecipitation detection, BMDMs plated in 6-well plates were stimulated to activate the NLRP3 inflammasome and lysed with NP-40 lysis buffer comprising complete protease inhibitor. The cell lysates were rotated and incubated overnight with the primary antibodies and protein A/G-agarose (Abmart, Shanghai, China) at 4°C. The antibody-bound proteins were precipitated with protein A/G beads, and immunoblot analysis was performed.

Sun T., Xie R., He H., Xie Q., Zhao X., Kang G., Cheng C., Yin W., Cong J., Li J, & Wang X. (2022). Kynurenic acid ameliorates NLRP3 inflammasome activation by blocking calcium mobilization via GPR35. Frontiers in Immunology, 13, 1019365.

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Verification of Nup96 Antibody Specificity

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The peptide (amino acid residues from 505 to 959) of Nup96 protein was used as the antigen to develop Nup96specific antibody by Abmart. To verify the specificity of Nup96 antibody, 12-d-old seedlings of Col-0, nup96-1, and nup96-2 were collected and ground to fine powder in liquid nitrogen. Three volumes of extraction buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, and 1× protease inhibitor mixture [Roche]) was added to the sample. After homogenization on ice for 20 min, the mixture was centrifuged at 13,000g at 4°C for 10 min. The supernatant was transferred to a new tube, 10% of which was saved as "input" sample. IP was performed as follows: 5 μL of Nup96 antibody was added to the cleared protein extracts. After incubation at 4°C for 3 h, 30 μL of Protein A/G-agarose (Abmart, A10001) was added and incubated for another 2 h at 4°C. After washing Protein A/G-agarose with extraction buffer (without protease inhibitor cocktail) five times, the proteins were eluted with 2× SDS-PAGE loading buffer by heating at 95°C for 5 min and separated on a 4 to 20% SurePAGE precast gel (Genscript, M00657) followed by immunoblot analyses.

Cheng Z., Zhang X., Huang P., Huang G., Zhu J., Chen F., Miao Y., Liu L., Fu Y.F, & Wang X. (2020). Nup96 and HOS1 Are Mutually Stabilized and Gate CONSTANS Protein Level, Conferring Long-Day Photoperiodic Flowering Regulation in Arabidopsis. The Plant cell, 32(2).

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