Cnbr activated sepharose 4b beads

Manufactured by GE Healthcare

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CNBr-activated Sepharose 4B beads are a type of agarose-based chromatography resin used for protein purification. They are designed to covalently immobilize ligands, such as enzymes or antibodies, for the capture and separation of target biomolecules from complex mixtures.

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CNBr-activated Sepharose (52 citations) Sepharose 4B beads (52 citations) CNBr-activated Sepharose 4B (45 citations) CNBr-Sepharose 4B beads (9 citations) CNBr-activated Sepharose beads (9 citations) CNBr-Sepharose (5 citations) CNBr-Sepharose beads (4 citations) CNBr‐Sepharose 4B (3 citations) -activated Sepharose™ 4B (2 citations) CNBr-beads (2 citations)

10 protocols using cnbr activated sepharose 4b beads

Conjugation of Mrk61 and W0-2 Antibodies

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Mrk61, a neo-epitope specific anti-sAPPβ rabbit monoclonal antibody (courtesy of MSD Research Laboratories), was generated and its specificity was previously characterized^{16 (link),17 (link)}. The purified antibody was conjugated with CNBr-activated Sepharose 4B beads (GE Healthcare) according to the manufacturer’s protocol, reconstituted into a 50% slurry of PBS containing 0.02% sodium azide and stored at 4 °C.
Purified W0-2 (courtesy of MSD Research Laboratories) was added to activated CNBr-Sepharose (1% w/w) pre-equilibrated in cold coupling buffer (0.1 M bicarbonate pH 7.6, 0.5 M NaCl) and incubated for 2 h with gentle mixing. Upon washing with coupling buffer, unoccupied binding sites were blocked for 2 h with 1 M ethanolamine, pH 8.0. Beads were washed with three cycles of 90 mL cold 0.1 M acetate pH 4.0 and 90 mL cold 0.1 M Tris pH 8.0 to remove uncoupled protein. The beads were further washed and stored (4 °C) in PBS containing 0.02% sodium azide.

Dobrowolska Zakaria J.A., Bateman R.J., Lysakowska M., Khatri A., Jean-Gilles D., Kennedy M.E, & Vassar R. (2022). The metabolism of human soluble amyloid precursor protein isoforms is quantifiable by a stable isotope labeling-tandem mass spectrometry method. Scientific Reports, 12, 14985.

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Esculetin Binding Affinity Assay

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CNBr-activated Sepharose 4B beads (GE Healthcare) were coupled with esculetin as described by Lee et al [24 (link)]. Coupled and uncoupled beads were separately incubated overnight with total cellular protein extract in reaction buffer [50 mM Tris (pH7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01 % Tergitol, 0.2 % BSA, 0.2 mM PMSF and 1X protease inhibitor cocktail]. Beads were washed thrice with reaction buffer followed by elution of retained protein in laemmli buffer with β mercaptoethanol. The protein was then fractionated on 10 % SDS-PAGE and checked for presence of KEAP1 by western blotting. Further, to assert the specificity of binding, competition assay was carried out by pre-incubating total cellular protein with variable concentrations of esculetin and subsequently using this protein mixture for pull down assay as described above.

Arora R., Sawney S., Saini V., Steffi C., Tiwari M, & Saluja D. (2016). Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1. Molecular Cancer, 15, 64.

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Affinity Purification of CXCR4 Receptors

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CNBr-activated Sepharose 4B beads (GE Healthcare) coupled to the rho1D4 monoclonal antibody (Cell Essentials, Boston, MA) were used to purify CXCR4 and CXCR4^QTY29 as previously described^{28 (link)}. Briefly, solubilized protein was captured on the beads overnight at 4 °C. The beads were washed with wash buffer (PBS + 0.01% or 0.2% FC14) until all impurities had been removed. The receptor was eluted from the beads with elution buffer (PBS + 0.01% or 0.2%FC14 + 800 µM elution peptide). The eluted protein was concentrated using 50 kDa MWCO (0.2% detergent samples), 30 kDa MWCO (0.2% or 0.01% detergent samples), or 10 kDa MWCO (0.01% detergent samples) filter columns. To remove excess elution peptide, the samples were washed with ~ 67 × volumes of wash buffer in the centrifugation filter columns at 4 ºC, 1000–1200 g, for intervals of 1–2 min. The centrifugation filter columns were from Millipore (Burlington, MA), and the elution peptide Ac-TETSQVAPA-CONH₂ was synthesized by CPC Scientific, Inc (Sunnyvale, CA).

Tegler L., Corin K., Pick H., Brookes J., Skuhersky M., Vogel H, & Zhang S. (2020). The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity. Scientific Reports, 10, 21371.

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Kinase Assay with Gossypin

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Gossypin (purity: > 90% by HPLC) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and CNBr-activated Sepharose 4B beads were from GE Healthcare (Piscataway, NJ, USA). The phosphorylation Aurora B (T232) antibody was obtained from Sigma-Aldrich and the total Aurora B antibody was from Abcam (Cambridge, MA, USA). The antibody to detect β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and all the other antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Active AURKA, active RSK2, MBP (AURKA substrate), and ATF1 (RSK2 substrate) human recombinant proteins for kinase assays were purchased from SignalChem (Richmond, BC, Canada).

Wang L., Wang X., Chen H., Zu X., Ma F., Liu K., Bode A.M., Dong Z, & Kim D.J. (2018). Gossypin inhibits gastric cancer growth by direct targeting AURKA and RSK2. Phytotherapy research : PTR, 33(3), 640-650.

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Synthesis and Characterization of PPMP

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We synthesized PPMP in-house by consulting a reported protocol and adding some modification [26 (link)]. Taxol was purchased from Selleck Chemicals (Houston, TX), and combretastatin A4 (CA4) was from Sigma-Aldrich Co. (St. Louis, MO). Basal Medium Eagle (BME), L-glutamine, gentamicin, penicillin, Eagle's Minimum Essential Medium (MEM), F-12K medium, and RPMI-1640 medium were all from Life Technologies, Inc. (Grand Island, NY). Fetal bovine serum (FBS) was obtained from Gemini Bio-Products (West Sacramento, CA). Primary antibodies against α-tubulin, β-actin or GAPDH were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and the other antibodies were purchased from Cell Signaling Technology (Danvers, MA) unless otherwise specified. CNBr-activated Sepharose^™ 4B beads were from GE Healthcare Biosciences (Pittsburgh, PA).

Sheng Y., Liu K., Wu Q., Oi N., Chen H., Reddy K., Jiang Y., Yao K., Li H., Li W., Zhang Y., Saleem M., Ma W.Y., Bode A.M., Dong Z, & Dong Z. (2016). PPMP, a novel tubulin-depolymerizing agent against esophageal cancer in patient-derived tumor xenografts. Oncotarget, 7(21), 30977-30989.

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Affinity Purification of Protein F991

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Affinity purified F991 was coupled to CNBr-activated sepharose 4B beads (17-0430-01, GE) for preparation of the affinity column. Purified F991 or control peptide (5 mg) was diluted in 6 ml coupling buffer (0.1 M NaHCO₃, 0.5 M NaCl, pH 8.3). Sepharose beads were then introduced to 1 mM HCl for 30 min and washed with coupling buffer. The F991 or control peptide (EGFRLSPGLG, synthesized by LifeTein LLC, Beijing, China) was added to resin, incubated overnight at 4 °C and blocked with blocking buffer (100 mM Glycine, pH 8.0) for 2 h at room temperature. F991-coupled sepharose and control peptide-coupled sepharose were transferred to the column and washed (3×) with alternating Tris-HCl buffer (0.1 M Tris-HCl buffer pH 8–9 containing 0.5 M NaCl) and acetate buffer (0.1 M acetate buffer pH 3–4 containing 0.5 M NaCl). Before use, these coupled columns were washed (3×) with PBS buffer (pH 7.2).

Ma J., Jiang D., Gong X., Shao W., Zhu Z., Xu W, & Qiu X. (2017). Free immunoglobulin light chain (FLC) promotes murine colitis and colitis-associated colon carcinogenesis by activating the inflammasome. Scientific Reports, 7, 5165.

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Production and Characterization of KIF13B Antibody

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An anti-KIF13B polyclonal antibody was produced by immunizing rabbits with a purified GST-tagged C-terminal KIF13B protein (1382–1843) expressed in BL21(DE3) Escherichia coli using the pGEX-4T-3 vector (GE Healthcare). The GST tag was removed from the protein by thrombin digestion before immunization. The antiserum was affinity purified using the antigen coupled to CNBr-activated Sepharose 4B beads (GE Healthcare). A rabbit polyclonal anti-KIF5B antibody, generated against the C-terminal sequence of KIF5B, was described previously (Kanai et al., 2000 (link)). Goat polyclonal anti-albumin (Bethyl Laboratories, Inc.), rat monoclonal anti-LAMP2 (ABL-93; Santa Cruz Biotechnology, Inc.), rabbit monoclonal anti-LRP1 (EPR3724; Abcam), rabbit monoclonal anti-LDLr (EP1553Y; Abcam), rabbit polyclonal anti-caveolin (sc-894; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-clathrin heavy chain (clone 23; BD), rabbit polyclonal anti-hDLG1 (sc-25661; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-utrophin (20C5, Abcam; 8A4, Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-myc (9B11; Cell Signaling Technology), and mouse monoclonal anti-FLAG (M2; Sigma-Aldrich) primary antibodies were purchased commercially. Alexa Fluor–conjugated secondary antibodies and Alexa Fluor 568–conjugated phalloidin were purchased from Invitrogen.

Kanai Y., Wang D, & Hirokawa N. (2014). KIF13B enhances the endocytosis of LRP1 by recruiting LRP1 to caveolae. The Journal of Cell Biology, 204(3), 395-408.

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Identification of Copine-III Interactors

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A chemically generated peptide consisting of nine amino acids (FTMEKGNRF) referred to the intracellular loop region of human EMP1 was immobilized onto CNBr-activated Sepharose 4B beads (GE Healthcare, Piscataway, NJ, USA). The beads coated with the peptide or uncoated were resuspended in LNCaP cell lysates for 16 h at 4 °C. Subsequently, proteins bound to the beads were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie Brilliant Blue staining for protein visualization. Following the excision of protein bands from the gel, they were destained and the proteins were digested by sequence-grade modified trypsin (Promega, Madison, WI, USA). The cleaved peptides were purified and analyzed by tandem mass spectrometry (Finigan LCQ Advantage MAX and Xcalibur Bioworks v.3.2; Thermo Electron, Waltham, MA, USA) to identify the protein sequences. Full-length human copine-III cDNA was subcloned into the pGEX-6P1 vector (GE Healthcare), and GST-copine-III was expressed in E. coli transformed with the vector. After sonicating the cells, GST-copine-III was purified from the cell lysates using glutathione-Sepharose 4B beads (GE Healthcare) as described previously [47 (link)].

Ahmat Amin M.K., Shimizu A., Zankov D.P., Sato A., Kurita S., Ito M., Maeda T., Yoshida T., Sakaue T., Higashiyama S., Kawauchi A, & Ogita H. (2018). Epithelial membrane protein 1 promotes tumor metastasis by enhancing cell migration via copine-III and Rac1. Oncogene, 37(40), 5416-5434.

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Preparation of SH2 Domain-Conjugated Sepharose Beads

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In brief, CNBr-activated Sepharose 4B beads (GE Healthcare) were washed with 1 mM HCl for 10 min to achieve activation for coupling. Purified SH2 domains that had been dialyzed into 0.1 M NaHCO₃ (pH 8.3) containing 0.5 M NaCl were added to the activated Sepharose beads. Beads were conjugated with excess protein (1 mg protein/0.04g of CNBr Sepharose) overnight at 4°C while rotating. Beads were then incubated with 100 mM Tris HCl (pH 8.0) for 2 h at room temperature to quench unreacted groups. Conjugated beads were washed with 3 cycles of alternating pH buffers [each cycle consists of 100 mM sodium acetate (pH 4.0) containing 0.5 M NaCl and then 0.1 mM Tris-HCl (pH 8.5) containing 0.5 M NaCl]. Finally, conjugated beads were washed and stored in PBS + 0.02% sodium azide at 4°C.

Crute B.W., Sheraden R., Ott V.L., Harley I.T., Getahun A, & Cambier J.C. (2020). Inhibitory Receptor Trap: A Platform for Discovery of Inhibitory Receptors That Utilize Inositol Lipid and Phosphotyrosine Phosphatase Effectors. Frontiers in Immunology, 11, 592329.

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Synthesis and Analysis of ADA-07

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ADA-07 was synthesized in-house and MS analysis was performed (Supplementary Figure 1A and B). Cell culture media were all obtained from Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) was from Gemini Bio-Products (West Sacramento, CA). Tris, NaCl, and SDS for molecular biology and buffer preparation were purchased from Sigma-Aldrich (St. Louis, MO). The active TOPK and MEK1 human recombinant proteins for the kinase assays were from SignalChem (Richmond, BC, Canada) and Millipore (Billerica, MA), respectively. The antibodies against phosphorylated TOPK (Thr9), ERK1/2 (Thr202/204), p38, JNKs, c-Jun and total TOPK, ERK1/2, p38, JNKs, p-c-Jun and PCNA were from Cell Signaling Biotechnology (Danvers, MA). Antibodies to detect β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit and the luciferase assay substrate were purchased from Promega (Madison, WI). CNBr-activated Sepharose™ 4B beads were purchased from GE Healthcare Bio-Sciences (Uppsala, Sweden).

Gao G., Zhang T., Wang Q., Reddy K., Chen H., Yao K., Wang K., Roh E., Zykova T., Ma W., Ryu J., Curiel-Lewandrowski C., Alberts D., Dickinson S.E., Bode A.M., Xing Y, & Dong Z. (2017). ADA-07 suppresses solar ultraviolet-induced skin carcinogenesis by directly inhibiting TOPK. Molecular cancer therapeutics, 16(9), 1843-1854.

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