Heparin-Sepharose (100 μl, Cytiva) was pre-equilibrated with PBS (Gibco) and then loaded with human FGF1 (Peprotech, #100-17A) or human FGF2 (Peprotech, #100-18B) dissolved in PBS, as previously described (65 (link)). The flow-through was reloaded onto the column twice to ensure complete binding. After washing twice with PBS, a 0.6 mg/ml solution of Sulfo-NHS-LC-biotin (Thermo Fisher) in PBS was loaded onto the column and incubated for 1 h at room temperature. Each column was washed three times with PBS, then bound biotinylated protein was eluted with 0.4 ml of PBS buffer containing an additional 2 M NaCl. All biotinylated proteins were stored at −80 °C.
Heparin sepharose
Manufactured by Cytiva
Sourced in United States
Heparin-Sepharose is a chromatography resin used for the purification and separation of proteins. It consists of heparin, a naturally occurring anticoagulant, immobilized on a cross-linked agarose matrix. The heparin ligand interacts with a wide range of proteins, allowing for the capture and separation of various biomolecules, including enzymes, growth factors, and coagulation factors.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (1 mentions)
Irdye 800, by Rockland Immunochemicals (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Precision plus protein dual colour standard, by Bio-Rad (1 mentions)
Goat serum, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Western blot detection system, by Thermo Fisher Scientific (1 mentions)
Protran, by Cytiva (1 mentions)
5 protocols using heparin sepharose
1
Biotinylation of FGF1 and FGF2
2
Affinity Purification of DR5-mTRAIL Complex
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Recombinant mouse DR5 (aa53-177)-Fc fusion protein (721-DR, R&D) alone (10 µg), or DR5–mTRAIL complex (10 µg each pre-incubated for 1 hr at room temperature), were loaded onto heparin-Sepharose (Cytiva) gravity column (200 µl bed volume). Column was first washed with 2 ml buffer A (25 mM HEPES, pH7.1, 150 mM NaCl), followed by four elution steps (800 µl each) using buffers containing 300 mM, 500 mM, 1 M, and 2 M NaCl, respectively. 30 µl of eluents from each step were resolved on a 4–20% SDS-PAGE gel and the gel was visualized by silver staining.
3
Prothrombin and ApoE Enrichment
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Primary antibodies used included a mouse monoclonal anti-prothrombin antibody (Catalog No. ab17199, Abcam) and a goat polyclonal anti-ApoE antibody (Catalog No. K74180B, Meridian Life Science). Secondary antibodies used were conjugated with either Alexa Fluor 680 (Invitrogen) or IRDye800 (Rockland) fluorophores for enhanced detection and visualization. Heparin-sepharose (Cytiva, lot#17099801 for Set 1 and lot#17099803 for Set 2) was used to enrich HBPs from plasma samples.
4
Optimized Techniques for TGase2 Assay and Detection
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Fluorescein-cadaverine (FC, Molecular ProbesTM A10466) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Putrescin, 4’,6-diamidino-2-phenylindole (DAPI), TGase assay kit (CS1070-1KT), RPMI 1640 medium, and goat serum were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Diethyl aminoethyl (DEAE) and Heparin Sepharose were purchased from Cytiva (Marlborough, MA, USA) and Sigma-Aldrich, respectively, and nitrocellulose membrane (Amersham Protran Supported) was purchased from GE Healthcare (Chicago, IL, USA). Human TGase 2 rabbit polyclonal antibodies (orb2986) were purchased from Biorbyt (Cambridge, UK). Horseradish-peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were purchased from Novus Biologicals Europe, UK. FITC-tagged anti-rabbit antibodies were obtained from Chemicon (Temecula, CA, USA). Fetal bovine serum (FBS, South America origin, EU Approved) was obtained from EuroClone, Italy. Protease inhibitor cocktail and the Western Blot detection system SuperSignal™ West Femto Maximum Sensitivity Substrate were obtained from Thermo Scientific (Waltham, MA, USA). Protein molecular mass markers ECL Plex Fluorescent Rainbow and Precision Plus Protein™ Dual Colour Standards were obtained from BIO-RAD (Hercules, CA, USA). All other chemicals were obtained from Sigma-Aldrich.
5
Affinity purification of DR5-mTRAIL complex
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Recombinant mouse DR5 (aa53-177)-Fc fusion protein (721-DR, R&D) alone (10μg), or DR5-mTRAIL complex (10 μg each pre-incubated for 1 h at room temperature), were loaded onto heparin-Sepharose (Cytiva) gravity column (200 μl bed volume). Column was first washed with 2 ml buffer A (25 mM HEPES, pH7.1, 150 mM NaCl), followed by four elution steps (800 μl each) using buffers containing 300 mM, 500mM, 1 M and 2M NaCl, respectively. 30 μl of eluents from each step were resolved on a 4-20% SDS-PAGE gel and the gel was visualized by silver staining.
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