Protein a sepharose

Manufactured by Thermo Fisher Scientific

Sourced in United States

Protein A-Sepharose is a chromatography medium used for the purification of antibodies and immunoglobulins. It is composed of Protein A, a bacterial cell wall protein, covalently coupled to Sepharose beads. Protein A has a high affinity for the Fc region of immunoglobulins, allowing for the selective capture and purification of antibodies from complex mixtures.

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Lab products found in correlation

35s methionine, by PerkinElmer (2 mentions) Mg132, by Merck Group (2 mentions) Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions) Flag m2 antibody, by Merck Group (2 mentions) Sera mag speedbead protein a g magnetic beads, by GE Healthcare (2 mentions) Isg15 antibody, by Thermo Fisher Scientific (2 mentions) X tremegene hp dna transfection reagent, by Roche (2 mentions) Protein a sepharose, by Sino Biological (2 mentions) Chaps cell extract buffer, by Cell Signaling Technology (2 mentions) Anti isg15 polyclonal, by Merck Group (2 mentions) Anti isg15 monoclonal, by Thermo Fisher Scientific (2 mentions) Sera mag speedbead protein a g, by GE Healthcare (2 mentions) Anti flag m2 antibody, by Merck Group (2 mentions) 35s easytag mix, by PerkinElmer (2 mentions) Methionine cysteine free dmem, by Thermo Fisher Scientific (2 mentions) Goat anti human iga agarose, by Merck Group (1 mentions) Tnt sp6 coupled reticulocyte lysate system, by Promega (1 mentions) Fk2 antibody, by Enzo Life Sciences (1 mentions) Nupage gradient gel, by Thermo Fisher Scientific (1 mentions) Rabbit anti mtor antibody, by Cell Signaling Technology (1 mentions)

44 protocols using protein a sepharose

Radioligand Binding Assay for tTG Autoantibodies

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A radioligand binding assay (RBA) were used to assess the changes in IgA-tTG and IgG-tTG levels (U/mL) separately, as previously described [31 (link)]. In short, human tissue transglutaminase (tTG) was synthesized by in vitro transcription and translation of cDNA using the TNT SP6 Coupled Reticulocyte Lysate System (Promega, Madison, WI, USA) in the presence of 35S-methionine (Perkin Elmer, Boston, MA, USA). Both IgG-tTG and IgA-tTG were analyzed. For the IgG-tTG analysis, 35S-tTG was diluted and added to human serum and incubated overnight at 4 °C. Protein A sepharose (PAS) (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) was used to separate free and antibody-bound 35S-tTG by binding IgG in the serum. The IgA-tTG analysis was performed similarly, except goat anti-human IgA-agarose (Sigma-Aldrich, St Louis, MO, USA) was used instead of the PAS. The levels of tTG autoantibodies were calculated from standard curves containing approximately 2 U/mL, 4 U/mL, 8 U/mL, 16 U/mL, 31 U/mL, 63 U/mL, 125 U/mL, 250 U/mL, 500 U/mL and 1000 U/mL of respective IgA-tTG and IgG-tTG.

Håkansson Å., Andrén Aronsson C., Brundin C., Oscarsson E., Molin G, & Agardh D. (2019). Effects of Lactobacillus plantarum and Lactobacillus paracasei on the Peripheral Immune Response in Children with Celiac Disease Autoimmunity: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial. Nutrients, 11(8), 1925.

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Signaling Protein Detection Protocol

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FK2 antibody was purchased from Enzo Life Sciences; antibodies against actin, PLCγ2 and Syk (N19 and 4D10) from Santa Cruz; LAT, SLP-76 from Millipore; PKCδ and PE-Cy5 anti-CD62P from BD Biosciences; phospho-specific antibodies for Syk (Y525/526, Y348, Y323) from Cell Signaling; and fluorescein isothiocyanate (FITC)-conjugated anti-fibrinogen antibody was from Agilent Technologies. PR619 and GST-Tandem Ubiquitin Binding Entities (GST-TUBEs) were from Tebu-Bio Ltd, and Protein A-Sepharose (PAS) and NuPage gradient gels from Invitrogen. Other reagents were from Sigma (Poole, United Kingdom).

Unsworth A.J., Bombik I., Pinto-Fernandez A., McGouran J.F., Konietzny R., Zahedi R.P., Watson S.P., Kessler B.M, & Pears C.J. (2018). Human Platelet Protein Ubiquitylation and Changes following GPVI Activation. Thrombosis and Haemostasis, 119(1), 104-116.

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Nrf2 Ubiquitination Assay in HEK 293 Cells

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HEK 293 cells transfected with plasmids that encode HA-Ub, V5-Nrf2, and Flag-Keap1, were treated with eCS (0.1 μg, 16 h), with or without MG132 (5 μM, 2 h). Nrf2 was precipitated with 1 μg of the anti-V5 antibody, the complex of which was pulled down with protein A-sepharose (Thermo Fisher Scientific) and immunoblotted with the anti-HA antibody for revealing the ubiquitinated Nrf2.

Han J.W., Kim K.H., Kwun M.J., Choi J.Y., Kim S.J., Jeong S.I., Lee B.J., Kim K.I., Won R., Jung J.H., Jung H.J, & Joo M. (2019). Suppression of lung inflammation by the ethanol extract of Chung-Sang and the possible role of Nrf2. BMC Complementary and Alternative Medicine, 19, 15.

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Coimmunoprecipitation of mTOR Complex

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For the coimmunoprecipitation experiments, cells were harvested in lysis buffer (40 mm HEPES, pH 7.5, 120 mm NaCl, 1 mm EDTA, 0.3% CHAPS, and protease and phosphatase inhibitors). The lysed cells were incubated on ice for 20 min and centrifuged for 10 min at 14,000 × g at 4°C to remove cellular debris. Supernatants that contained 1.5 mg total protein were then incubated with rabbit anti-mTOR antibody (Cell Signaling Technology, catalogue no. 2972) at 4°C overnight, and then Protein A Sepharose (Thermo Fisher Scientific, catalogue no. 101041) was added. As a negative control, normal rabbit immunoglobulin G (IgG; Sigma, catalogue no. 12-370) was used. After incubation for 2 h at 4°C, the resins were washed four times with lysis wash buffer and boiled in SDS–PAGE sample buffer. The samples were analyzed by Western blot.

Samluk L., Urbanska M., Kisielewska K., Mohanraj K., Kim M.J., Machnicka K., Liszewska E., Jaworski J, & Chacinska A. (2019). Cytosolic translational responses differ under conditions of severe short-term and long-term mitochondrial stress. Molecular Biology of the Cell, 30(15), 1864-1877.

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Evaluating Nrf2 Ubiquitination in HEK293 Cells

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Ubiquitination assay was performed as described elsewhere [23 (link)]. In brief, HEK293 cells were transfected with plasmids expressing HA-Ub, V5-Nrf2, and FLAG-Keap1 for 48 h. Prior to lysis, cells were treated with 10 μM of MG132 for 3 h. V5-Nrf2 was precipitated by anti-V5 antibody, captured by protein A-sepharose (Thermo Scientific), and analyzed by immunoblotting for HA-Ubiquitin to reveal the ubiquitinated Nrf2.

Kim K.H., Ahn S., Won R., Lee J.J., Kim T.H., Kim J.I., Choi J.Y, & Joo M. (2020). Sikyungbanha-Tang Suppressing Acute Lung Injury in Mice Is Related to the Activation of Nrf2 and TNFAIP3. Evidence-based Complementary and Alternative Medicine : eCAM, 2020, 8125758.

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Ubiquitination Assay for Nrf2 Regulation

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Ubiquitination assay was performed as described elsewhere [20 (link)]. In brief, HEK293 cells (ATCC) were transfected with plasmids expressing HA-tagged Ub, V5-tagged Nrf2, and FLAG-tagged Keap1 [21 (link)] for 48 h and then treated with MGS for 16 h. Prior to lysis, cells were treated with 10 μM of MG132 (Sigma-Aldrich) for 3 h to block ubiquitin-dependent protein degradation. The total cell lysate was prepared by Pierce™ IP lysis buffer per the manufacturer’s protocols (Thermo Scientific). For precipitating V5-tagged Nrf2, 1 μg of anti-V5 antibody (R960–25, Thermo Scientific) was added to the cytosolic fraction. Immune complexes captured by protein A-sepharose (Thermo Scientific) were analyzed by immunoblotting for HA (H3663, Sigma) to reveal the ubiquitinated Nrf2.

Kim K.H., Lee J.Y., Ahn S., Won R., Kim S.J., Jeong S.I., Lee J.J., Kim J.I., Choi J.Y, & Joo M. (2020). The methanol extract of Guettarda speciosa Linn. Ameliorates acute lung injury in mice. BMC Complementary Medicine and Therapies, 20, 40.

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Characterization of Antibody Reagents

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The mAbs hec1 (anti-VE-cadherin), hec7 (anti-PECAM-1), and hec2 (anti-CD99) were produced from hybridomas generated in the laboratory as previously described (32 (link)). P1.1 (non-blocking anti-PECAM mAb) was column purified on protein A-Sepharose (GE Healthcare) from ascites provided by P.J. Newman (Blood Center of Wisconsin, Milwaukee, WI) (33 (link)). Fab fragments of P1.1 were cut using papain (Thermo Fisher Scientific), followed by column purification on protein A-Sepharose. Purity of Fab fragments was confirmed by SDS-PAGE. Unlabeled and Dylight 549 conjugated goat-anti-mouse F(ab’)₂ antibodies used for targeted recycling experiments of PECAM-1 were purchased from Jackson ImmunoResearch laboratories. Antibodies for immunofluorescence were conjugated to DyLight 488, DyLight 550 or Alexa Fluor 650 according to the manufacturers protocol (Thermo Fisher Scientific). Alexa Fluor 488 conjugated monoclonal antibodies against occludin and claudin-5 were purchased from Life Technologies (Invitrogen). Polyclonal antibody against claudin-3 was purchased from Life Technologies (Invitrogen). 8-(4-chlorophenylthio(CPT)-cAMP was purchased from Sigma Aldrich and phosphodiesterase inhibitor type IV RO-20–1724 was purchased from Tocris Bioscience.

Winger R.C., Koblinski J.E., Kanda T., Ransohoff R.M, & Muller W.A. (2014). Rapid Remodeling of Tight Junctions During Paracellular Diapedesis in a Human Model of the Blood-Brain Barrier. Journal of immunology (Baltimore, Md. : 1950), 193(5), 2427-2437.

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Immunoprecipitation of Cellular Proteins

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HeLa and HEK293T cells were transfected as described above. Briefly, 1 mg of protein was immunoprecipitated with agarose-conjugated anti-GFP beads for 2 h as described by the manufacturer (MLB). For RFP IP, 1mg of protein was immunoprecipitated with magnetic anti-RFP beads for 2 h as described by the manufacturer (MLB). For p24 IP, 1mg of protein was pre-cleared with normal mouse or rabbit serum and 25 μl of a 50:50 slurry of protein A-Sepharose or protein G-Sepharose (Thermo Scientific), incubated with mouse (NIH AIDS Reference and Reagent Program) or rabbit (Fritzgerald) anti-p24 (American BioTechnologies; dilution 1:200) for 16 h at 4°C, and with 30 μl of 50:50 slurry of protein A-Sepharose or protein G-Sepharose for 2 h at 4 °C. For Rluc IP, 1mg of protein was incubated with rabbit anti-Rluc (MBL) for 16 h at 4°C, and with 30 μl of 50:50 slurry of protein A-Sepharose for 2 h at 4°C. The bound complexes were analyzed by SDS-PAGE analysis.

Valiente-Echeverría F., Melnychuk L., Vyboh K., Ajamian L., Gallouzi I.E., Bernard N, & Mouland A.J. (2014). eEF2 and Ras-GAP SH3 domain-binding protein (G3BP1) modulate stress granule assembly during HIV-1 infection. Nature communications, 5, 4819.

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Fluorescent Labeling of Anti-ROR1 Antibodies

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Clone F anti-ROR1 antibodies were expressed in full length human IgG1 format. Heavy chain and light chain sequences were ordered as gblock gene fragments (IDT) and individually cloned into the cytomegalovirus-driven adenoviral shuttle vector pAdd2 using standard Gibson assembly. Cloned expression vectors were transfected into Expi293 cells (Thermo Fisher, A14526) in a 1:1 weight ratio of heavy chain to light chain and expressed according to manufacturer’s protocol. Antibody was purified from the supernatant using protein A affinity chromatography (Protein A Sepharose, Thermo Fisher, 101041). The purified antibody was fluorescently labeled with the DyLight 650 Microscale Antibody Labeling Kit (Thermo Fisher).

Labanieh L., Majzner R.G., Klysz D., Sotillo E., Fisher C.J., Vilches-Moure J.G., Pacheco K.Z., Malipatlolla M., Xu P., Hui J.H., Murty T., Theruvath J., Mehta N., Yamada-Hunter S.A., Weber E.W., Heitzeneder S., Parker K.R., Satpathy A.T., Chang H.Y., Lin M.Z., Cochran J.R, & Mackall C.L. (2022). Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell, 185(10), 1745-1763.e22.

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ChIP, EMSA, and Western Blot Protocols

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Western blots were performed according to standard procedures. ChIP and electrophoretic mobility shift assay (EMSA) were performed as described (26 (link)). For BATF the ChIP method was adapted such that protein A Sepharose (Thermo Scientific) was first saturated with rabbit anti-mouse secondary antibody, and then incubated with anti-BATF or control mouse IgG. Pre-bound beads were used to immunoprecipiate chromatin fractions. Nuclear extracts for EMSA were prepared from transfected COS cells, and OCI-LY3 and -LY10 cell lines. For EMSA probe sequences and ChIP PCR primer sequences see Supplementary Methods.

Care M.A., Cocco M., Laye J.P., Barnes N., Huang Y., Wang M., Barrans S., Du M., Jack A., Westhead D.R., Doody G.M, & Tooze R.M. (2014). SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity. Nucleic Acids Research, 42(12), 7591-7610.

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