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Immobilized protein a g beads

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Immobilized protein A/G beads are a type of affinity chromatography resin used for the purification of immunoglobulins (Ig) and other proteins that bind to protein A or protein G. The beads consist of protein A or protein G covalently coupled to a cross-linked agarose matrix, providing a high-capacity support for the capture and isolation of target proteins from complex samples.

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Lab products found in correlation

Ubiquitin, by Cell Signaling Technology (3 mentions) Leupeptin, by Merck Group (3 mentions) Control igg, by Santa Cruz Biotechnology (3 mentions) Escherichia coli top 10 competent cells, by Thermo Fisher Scientific (3 mentions) Bleomycin, by Merck Group (2 mentions) Smad2 3, by Cell Signaling Technology (2 mentions) Mammalian expressional plasmid pcdna3.1 his v5 topo, by Thermo Fisher Scientific (2 mentions) P smad2 3, by Cell Signaling Technology (2 mentions) β actin antibody, by Merck Group (2 mentions) Cycloheximide (chx), by Merck Group (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) V5 antibody, by Thermo Fisher Scientific (2 mentions) Emem media, by Thermo Fisher Scientific (1 mentions) Usp11, by Santa Cruz Biotechnology (1 mentions) V5 tag, by Santa Cruz Biotechnology (1 mentions) Tβrii, by Santa Cruz Biotechnology (1 mentions) Antibodies against flag tag and β actin, by Merck Group (1 mentions) Recombinant tgf β1, by Thermo Fisher Scientific (1 mentions) Ha tag, by Santa Cruz Biotechnology (1 mentions) Total smad2, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Protein A/G (60 citations) Protein G beads (41 citations) Protein A beads (27 citations) G/A beads (2 citations)

7 protocols using immobilized protein a g beads

1

AJAP1 Co-Immunoprecipitation Assay

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Cells were treated with MG132 (20 μM) for 8 h, harvested, and lysed in 400 μl co-IP buffer containing a protease inhibitors cocktail (Sigma-Aldrich). The lysate was centrifuged and the supernatant was precleared by incubation with 20 μl immobilized protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 4 °C, followed by overnight incubation at 4 °C with 20 μl anti-AJAP1 antibody or control IgG. Protein complexes were precipitated by incubation with 30 μl immobilized protein A/G beads for 2 h at 4 °C. Samples were washed four times with lysis buffer; the beads were boiled in loading buffer, and proteins were detected by western blotting.
Han J., Xie C., Pei T., Wang J., Lan Y., Huang K., Cui Y., Wang F., Zhang J., Pan S., Liang Y., Zhen T., Song R., Sun B., Li Y., Shi H., Yang G., Liu X., Zhu M., Wang Y., Li K., Liu Y., Meng F., Liao F., Meng X., Hong X, & Liu L. (2017). Deregulated AJAP1/β-catenin/ZEB1 signaling promotes hepatocellular carcinoma carcinogenesis and metastasis. Cell Death & Disease, 8(4), e2736-.
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2

Fibroblast Cell Culture and TGF-β Signaling

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Human fetal lung fibroblast (MRC5) cell lines were propagated in EMEM media (Gibco, Rockville, IL, USA) with 10% FBS (Hyclone, Logan, UT, USA), and 1% penicillin/streptomycin antibiotic mix (Lonza, Allendale, NJ, USA). The cells were kept in a 37 °C humidified incubator with 5% carbon dioxide. Immobilized protein A/G beads, FN, α-SMA, TβRI, HA tag, USP11, V5 tag, TβRII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-SMAD2, phospho-SMAD3, total SMAD2, SMAD3, and ubiquitin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Bleomycin, leupeptin, CHX, MTX, and antibodies against Flag-tag and β-actin were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TGF-β1 was purchased from Invitrogen (Carlsbad, CA, USA). Proteasome inhibitor MG-132 was from Calbiochem (KGaA, Darmstadt, Germany). DAPI was purchased from ThermoFisher Scientific (Waltham, MA, USA). All materials in highest grades used in the experiments are commercially available.
Jacko A.M., Nan L., Li S., Tan J., Zhao J., Kass D.J, & Zhao Y. (2016). De-ubiquitinating enzyme, USP11, promotes transforming growth factor β-1 signaling through stabilization of transforming growth factor β receptor II. Cell Death & Disease, 7(11), e2474-.
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3

Lung Fibroblast Cell Culture Protocol

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Human lung fibroblast cell lines (Mrc5 and IMR90) were purchased from ATCC (Manassas, VA). Human primary lung fibroblasts (HLF) and lung myofibroblasts (IPF-LF) from adult normal human subjects and IPF patients were obtained from the Center for Organ Recovery and Education and Lung Transplantation at the University of Pittsburgh. The study was approved by the institutional Review Board at the University of Pittsburgh (STUDY18100070). Cells were cultured in Eagle’s Minimum Essential Medium (EMEM) containing 10% fetal bovine serum (FBS) in 5% CO2 cell culture incubator. V5 antibody, mammalian expressional plasmid pcDNA3.1/His-V5-topo, and Escherichia coli Top10 competent cells were purchased from Life technologies (Grand Island, NY). Nedd4L, TβRII, collagen I, ubiquitin, smurf1, Smad4, p-Smad2/3, and Smad2/3 antibodies were purchased from Cell Signaling (Danvers, MA). Fibronectin (FN), alpha-smooth muscle actin (α-SMA), E2F4 antibodies, immobilized protein A/G beads, and control IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HLM006474, bleomycin, and β-actin antibody were purchased from Sigma (St. Louis, MO). Human recombinant TGF-β1 was purchased from R&D systems (Minneapolis, MN). All materials used in the experiments are the highest grade commercially available.
Li S., Ye Q., Wei J., Taleb S.J., Wang H., Zhang Y., Kass D.J., Horowitz J.C., Zhao J, & Zhao Y. (2022). Nedd4L suppression in lung fibroblasts facilitates pathogenesis of lung fibrosis. Translational research : the journal of laboratory and clinical medicine, 253, 1-7.
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4

Investigating Sigirr Regulation via USP13

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Mouse lung epithelial cells (MLE12) and Human monocyte-like cell line (THP1) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Recombinant human IL-37 was from R&D Systems (Minneapolis, MN, USA). Cycloheximide, leupeptin, and LPS were purchased from Sigma Aldrich (St Louis, MO, USA). MG-132 was purchased from EMD Chemicals (Gibbstown, NJ, USA). Immobilized protein A/G beads and control IgG and anti-Sigirr (for mouse, rat, and human origin) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-USP13 was purchased from Protein Tech (Chicago, IL, USA) and Bethyl Laboratories (Montgomery, TX, USA). Antibodies against HA tag, GSK3β, pY216GSK3β, phospho-threonine, and ubiquitin were purchased from Cell Signaling (Beverly, MA, USA). Anti-V5, mammalian expression plasmid pcDNA3.1/His-V5-topo, Escherichia coli Top10 competent cells were purchased from Invitrogen and Thermo life technology (Gaithersburg, Maryland, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Bio-Rad Laboratories (Hercules, CA, USA). All commercially available materials used were of the highest quality.
Li L., Wei J., Suber T.L., Ye Q., Miao J., Li S., Taleb S.J., Tran K.C., Tamaskar A.S., Zhao J, & Zhao Y. (2021). IL-37-induced activation of glycogen synthase kinase 3β promotes IL-1R8/Sigirr phosphorylation, internalization, and degradation in lung epithelial cells. Journal of cellular physiology, 236(8), 5676-5685.
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5

Epithelial Cell Culture and Protein Analysis

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Human bronchial epithelial cell line (HBE2) was cultured with HITES medium containing 10% fetal bovine serum (FBS). HEK293 cells were cultured with Dulbecco’s Modified Eagle Medium containing 10% FBS. Human lung fibroblast cells (Mrc5) were cultured with Eagle’s Minimum Essential Medium containing 10% FBS. V5 antibody, pcDNA3.1/His V5 TOPO plasmid and Escherichia coli top 10 competent cells were from Invitrogen (Carlsbad, California, USA). MG-132 was from Calbiochem (La Jolla, California, USA). Leupeptin, cycloheximide and β-actin antibody were from Sigma-Aldrich (St. Louis, Missouri, USA). Immobilized protein A/G beads, ubiquitin antibody and ISG15 antibody were from Santa Cruz Biotechnology (Santa Cruz, California, USA). Smad2/3, p-Smad2/3, fibronectin, smooth muscle actin and FOXO3a antibodies were from Cell Signaling Technology (Danvers, Massachusetts, USA). All materials used in the experiments were in the highest grades commercially available.
Wang B., Li Y., Wang H., Zhao J., Zhao Y., Liu Z, & Ma H. (2019). FOXO3a is stabilized by USP18-mediated de-ISGylation and inhibits TGF-β1-induced fibronectin expression. Journal of Investigative Medicine, 68(3), 786-791.
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6

Cell-Based Assay for Endothelial Barrier

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Human lung microvascular endothelial cells (HLMVECs), THP-1 cells, MLE12 cells, and Raw 264.7 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HLMVECs were cultured with EGM-2MV microvascular endothelial growth medium containing 5% fetal bovine serum (FBS) at 37 °C in 5% CO2. LPS (E. coli O55:B5), thrombin, and the anti-β-actin antibody were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies against VE-cadherin, USP40, ICAM1, VCAM1, Lamin A/C, and GAPDH and immobilized protein A/G beads were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against pMLC, MLC, the Flag tag, the V5 tag, pLimk1, Limk1, p-cofilin 1, cofilin 1, pIκBα, p65, p-p65, HSP90β, K63-linked ubiquitin, ubiquitin, and acetylated lysine (AcK) were purchased from Cell Signaling (Beverly, MA, USA). ELISA kits for quantifying TNFα, IL-6, and IL-8 were obtained from eBioscience (San Diego, CA), and mouse IL-1β, KC/CXCL1 ELISA kits, and human recombinant TNFα protein were purchased from R&D Systems (Minneapolis, MN, USA). The anti-CD31-FITC antibody was obtained from Biolegend (San Diego, CA, USA). GeneJet™ reagent and GeneMute siRNA transfection reagent were purchased from SignaGen (Frederick, MD, USA). All materials used in the experiments were of the highest grade commercially available.
Miao J., Li L., Shaheen N., Wei J., Jacko A.M., Sundd P., Taleb S.J., Mallampalli R.K., Zhao Y, & Zhao J. (2024). The deubiquitinase USP40 preserves endothelial integrity by targeting the heat shock protein HSP90β. Experimental & Molecular Medicine, 56(2), 395-407.
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7

Cultured Human Lung Endothelial Cells

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Human lung microvascular endothelial cells (HLMVECs, from ATCC) were cultured with endothelial growth medium-2 (EGM-2) containing antibiotics at 37°C in 5% CO2. Human hybridoma endothelial cells (EAhy 926, ATCC) and HEK293 cells (from ATCC) were cultured in DMEM medium containing 10% fetal bovine serum (FBS). V5 tag antibody, mammalian expressional plasmid pcDNA3.1/His-V5-topo, and Escherichia coli Top10 competent cells were purchased from Life technologies (Grand Island, NY). ISG15, p65, ubiquitin, and WIP1 antibodies were purchased from Cell Signaling (Danvers, MA). ICAM1 and VCAM1 antibodies, immobilized protein A/G beads, and control IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FBXL19 antibody was from Abgent (San Diego, CA). Lipopolysaccharide (LPS), T7 tag, and β-actin antibodies were purchased from Sigma (St. Louis, MO). Human recombinant TNFα was purchased from R&D systems (Minneapolis, MN). All materials used in the experiments are the highest grade commercially available.
Li L., Miao J., Shaheen N., Taleb S.J., Hu J., Ye Q., He J., Yan J., Mallampalli R.K., Zhao J, & Zhao Y. (2023). ISGylation of NF-κBp65 by SCFFBXL19 E3 ligase diminishes endothelial inflammation. Arteriosclerosis, thrombosis, and vascular biology, 43(5), 674-683.
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