Ech sepharose 4b

Manufactured by GE Healthcare

Sourced in United States

ECH Sepharose 4B is a lab equipment product used for affinity chromatography. It is a cross-linked agarose-based bead that can be used to purify and separate biomolecules such as proteins, enzymes, and antibodies from complex mixtures. The product provides a stable and porous matrix for immobilizing ligands, enabling efficient capture and separation of target molecules.

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Lab products found in correlation

Purvalanol b, by Bio-Techne (2 mentions) Ctx 0294885, by GE Healthcare (2 mentions) Eah sepharose 4b, by GE Healthcare (2 mentions) Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Eah sepharose 4b beads, by GE Healthcare (1 mentions) Carboplatin, by Selleck Chemicals (1 mentions) A 674563, by Selleck Chemicals (1 mentions) Bis acrylamide, by Bio-Rad (1 mentions) Bio safe coomassie g 250 stain, by Bio-Rad (1 mentions) Precision plus protein dual xtra standard, by Bio-Rad (1 mentions) Laemmli sample buffer, by Bio-Rad (1 mentions) Ouabain, by Merck Group (1 mentions) Acrylamide, by Bio-Rad (1 mentions) Silver stain plus kit, by Bio-Rad (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Sequencing grade modified trypsin, by Promega (1 mentions) Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions) Readyprep 2 d cleanup kit, by Bio-Rad (1 mentions)

Close spelling variants of this product

Sepharose 4B (96 citations) ECH sepharose 4B beads (2 citations)

6 protocols using ech sepharose 4b

Kinome Profiling of Breast Cancer Cells

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Breast tumors harvested for kinome/MIB-assay studies were homogenized before protein extraction. Additionally, protein extracts were collected from 67NR cells (1.5x10⁶) grown for 24 hours before treatment with AEE788 (1 μM), ATX-101 (6 μM) or the combination of these for additional 24 hours (n=3 for each treatment group). Proteins were extracted using Mammalian Protein Extraction Reagent (Thermo Fischer Scientific) according to the manufacturer’s instructions and with Halt Protease inhibitor cocktail (EDTA free) and Halt Phosphatase inhibitor cocktail (Thermo Fischer Scientific) added. The final protein concentrations were adjusted to 1 mg/mL.
The MIB-assay enriching for kinases was performed using three different kinase inhibitors (Purvalanol B (Tocris Bioscience), Bisindolylmaleimide X (Activate Scientific) and SB6-060-05 [49 (link)]) immobilized on ECH sepharose 4B and EAH sepharose 4B beads (GE healthcare) as described [29 (link)], using 200 μL protein extract (1 mg/mL) per column with beads. Proteins in the eluates were identified using mass spectrometry (MS)-analysis (Orbitrap) as described [29 (link)].

Søgaard C.K., Nepal A., Petrovic V., Sharma A., Liabakk N.B., Steigedal T.S, & Otterlei M. (2019). Targeting the non-canonical roles of PCNA modifies and increases the response to targeted anti-cancer therapy. Oncotarget, 10(68), 7185-7197.

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Compound Synthesis and Immobilization

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A-674563, Carboplatin, FRAX1036, and PF-03758309 were purchased from Selleckchem. BDP5290 and BDP9066 were purchased from Glixx Labs and ProbeChem respectively (Data file S10). For the compounds utilized in MIB synthesis, Purvalanol B was purchased from Abcam. PP58 (42 (link)) and VI16832 (43 (link)) were custom synthesized according to previously described methods by The Center for Combinatorial Chemistry and Drug Discovery, Jilin University, P.R. China. CTx-0294885 (44 (link)) was purchased from MedKoo Biosciences, Inc (406457). Conjugation of inhibitors to beads was performed by carbodiimide coupling to ECH Sepharose 4B (CTx-0294885, VI16832 and PP58) or EAH Sepharose 4B (purvalanol B) (GE Healthcare).

Kurimchak A.M., Herrera-Montavez C., Brown J., Johnson K.J., Sodi V., Srivastava N., Kumar V., Deihimi S., O’Brien S., Peri S., Mantia-Smaldone G.M., Jain A., Winters R.M., Cai K.Q., Chernoff J., Connolly D.C, & Duncan J.S. (2020). Functional proteomics interrogation of the kinome identifies MRCKA as a therapeutic target in high-grade serous ovarian carcinoma. Science signaling, 13(619), eaax8238.

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Kinobead Affinity Reagent Synthesis and Immobilization

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The kinobead affinity reagents (Fig. 1a) as well as the inhibitors 1553, 1561, 1649 and 1369 (Fig. 4a) were synthesized in-house. The kinobead affinity reagents were immobilized on ECH Sepharose 4B (GE Healthcare Bio-Sciences, Pittsburgh, PA) according to the previously published protocol.^{24 (link)}

Golkowski M., Vidadala R.S., Lombard C.K., Suh H.W., Maly D.J, & Ong S.E. (2017). Kinobead and single-shot LC-MS profiling identifies selective PKD inhibitors. Journal of proteome research, 16(3), 1216-1227.

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Kinase Inhibitor Purification and Assay

Cited 1 time

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Three different kinase inhibitors (Purvalanol B (Tocris Bioscience), Bisindolmaleimide X (Activate Scientific), and SB6-060-05 [30 (link)]) were immobilized using ECH Sepharose 4B and EAH Sepharose 4B (GE Healthcare) beads, according to the manufacturer’s instructions and as published elsewhere [31 (link)]. The following steps were performed as described [32 (link)], using 100 µL (0.1 mg) of cell extract per column (50 µL of mixed inhibitor beads).

Petrovic V., Nepal A., Olaisen C., Bachke S., Hira J., Søgaard C.K., Røst L.M., Misund K., Andreassen T., Melø T.M., Bartsova Z., Bruheim P, & Otterlei M. (2018). Anti-Cancer Potential of Homemade Fresh Garlic Extract Is Related to Increased Endoplasmic Reticulum Stress. Nutrients, 10(4), 450.

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Purification and Analysis of Neurotransmitters

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Sequencing Grade Modified Trypsin was purchased from Promega Corporation (Madison, WI, USA). ECH Sepharose 4B was purchased from GE (Fairfield, CT, USA). Pierce® BCA Protein Assay Kit was purchased from Thermo Fisher Scientific Inc. (MA, USA). Bio-Scale MT Columns, acrylamide, Bis-acrylamide, ReadyPrep™ 2-D Cleanup Kit, Silver Stain Plus Kit, Bio-Safe Coomassie Stain G250, Precision Plus Protein™ Dual Xtra Standards and Laemmli sample buffer were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). mMESSAGE mMACHINE® T7 Transcription Kit was purchased from Life Technologies (Carlsbad, CA, USA). Clark Borosilicate Thin Wall with Filament GC150TF were purchased from Warner Instruments, Inc. (Hamden, CT, USA). Formic acid (FA), acetonitrile (ACN), acetylcholine (ACh), imidacloprid (Imi), dihydro-β-erythroidine (dHβE) and ouabain were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).
Nitenpyram was generously provided by Professor Zhong Li of East China University of Science and Technology.

Bao H., Sun H., Xiao Y., Zhang Y., Wang X., Xu X., Liu Z., Fang J, & Li Z. (2015). Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen). Scientific Reports, 5, 8849.

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Inhibitor Conjugation and Characterization

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BIX02189, bortezomib, GDC-0941, trametinib, LY3009120, and sorafenib were purchased from Selleckchem, and JQ1 was purchased from ApexBio. PP58 (15 (link)) and VI16832 (16 (link)) were custom synthesized according to previously described methods by The Center for Combinatorial Chemistry and Drug Discovery, Jilin University, P.R. China. CTx-0294885 (17 (link)) was purchased from Medkoo. Conjugation of inhibitors to beads was performed by carbodiimide coupling to ECH Sepharose 4B (CTx-0294885, VI16832 and PP58) or EAH Sepharose 4B (purvalanol B) (GE Healthcare).

Kurimchak A.M., Shelton C., Herrera-Montávez C., Duncan K.E., Chernoff J, & Duncan J.S. (2019). Intrinsic Resistance to MEK Inhibition Through BET Protein Mediated Kinome Reprogramming in NF1-deficient Ovarian Cancer. Molecular cancer research : MCR, 17(8), 1721-1734.

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