Thermo Fisher Scientific EDC User Guide
PDF
Download
Document
Advertisement
Advertisement
INSTRUCTIONS EDC MAN0017125 Rev. A.0 Pub. Part No. 2160475.7 22980 22981 77149 Number Description 22980 EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), 5g 22981 EDC, 25g 77149 EDC, 10mg N Molecular Weight: 191.7 CAS # 25952-53-8 NH Cl C N Storage: Upon receipt store desiccated at -20°C. Products are shipped with an ice pack. For Research Use Only. Not for use in diagnostic procedures. Introduction Thermo Scientific™ EDC is a carboxyl and amine-reactive zero-length crosslinker. EDC reacts with a carboxyl group first and forms an amine-reactive O-acylisourea intermediate that quickly reacts with an amino group to form an amide bond and release of an isourea by-product (see the Additional Information Section). The intermediate is unstable in aqueous solutions, and therefore, two-step conjugation procedures rely on N-hydroxysuccinimide for stabilization.1,2 Failure to react with an amine will result in hydrolysis of the intermediate, regeneration of the carboxyl and release of an N-substituted urea. A side reaction is the formation of an N-acylurea, which is usually restricted to carboxyls located in hydrophobic regions of proteins.1,3 Procedure for Using EDC for Coupling Haptens to a Carrier Protein Materials Required • Carrier protein: 2mg bovine serum albumin (BSA), ovalbumin (OVA) or keyhole limpet hemocyanin (KLH) • Conjugation Buffer: 0.1M MES (2-[N-morpholino]ethane sulfonic acid), pH 4.5-5 (Product No. 28390) • • • EDC: 10mg Hapten: 1-2mg Thermo Scientific™ Zeba™ Spin Desalting Column (Product No. 89891) or other gel filtration column with a 5-6K molecular-weight cutoff Procedure 1. Equilibrate EDC to room temperature. 2. Add 2mg of lyophilized BSA, OVA or KLH to 200µL Conjugation Buffer. If using Thermo Scientific™ Imject™ Carrier Proteins, reconstitute using ultrapure water. 3. Dissolve up to 2mg of the peptide or hapten in 500µL of Conjugation Buffer and add it to the 200µL carrier protein solution. 4. For BSA or OVA conjugation, dissolve 10mg of EDC in 1mL of ultrapure water and immediately add 100µL of this solution to the carrier-peptide solution. For KLH conjugation, dissolve 10mg of EDC in 1mL of ultrapure water and immediately add 50µL of this solution to the carrier-peptide solution. Further reduce the amount of EDC if precipitation occurs. React for 2 hours at room temperature. 5. 6. Purify the conjugate using a desalting column. If storing the immunogen for more than a few days, sterile filter the conjugate and store in a sterile container at 4°C or -20°C. Pierce Biotechnology 3747 N. Meridian Road PO Box 117 Rockford, lL 61105 USA (815) 968-0747 (815) 968-7316 fax Thermofisher.com Procedure for Two-step Coupling of Proteins Using EDC and NHS or Sulfo-NHS The following protocol, adapted from a procedure described by Grabarek and Gergely, allows sequential coupling of two proteins without affecting the second protein’s carboxyls by exposing them to EDC. This procedure requires quenching the first reaction with a thiol-containing compound. The activation reaction with EDC and Sulfo-NHS is most efficient at pH 4.5-7.2; however, the reaction of NHS-activated or Sulfo-NHS-activated molecules with primary amines is most efficient at pH 7-8. For best results, perform the first reaction in MES buffer (or other non-amine, non-carboxylate buffer) at pH 5-6, then raise the pH to 7.2-7.5 with phosphate buffer (or other non-amine buffer) immediately before reaction to the amine-containing molecule. For quenching the first reaction, use 2-mercaptoethanol, or the excess reagent can be simply removed (as well as the reaction pH adjusted) by buffer-exchange with a desalting column. Materials Required • • • • • • • • Activation Buffer: 0.1M MES, 0.5M NaCl, pH 6.0 Coupling Buffer: Phosphate-buffered saline (PBS), 100mM sodium phosphate, 150mM NaCl; pH 7.2 (Product No. 28372) Protein # 1: Prepared in Activation Buffer at 1mg/mL Protein # 2: Prepared in Coupling Buffer NHS or Sulfo-NHS (Product No. 24500 and 24510, respectively) 2-Mercaptoethanol (Product No. 35600) (Optional) Zeba Spin Desalting Column (Product No. 89891) or other gel filtration column Hydroxylamine•HCl (Product No. 26103) Procedure 1. Equilibrate EDC and NHS to room temperature before opening bottles. 2. Add 0.4mg EDC (~2mM) and 0.6mg of NHS or 1.1mg of sulfo-NHS (~5mM) to 1mL of protein #1 solution and react for 15 minutes at room temperature. 3. Add 1.4µL of 2-mercaptoethanol (final concentration of 20mM) to quench the EDC. 4. Optional: Separate the protein from excess reducing agent and inactivated crosslinker using a desalting column that has been equilibrated with Coupling Buffer (PBS). 5. Add protein #2 to the activated protein at an equal molar ratio with protein #1. Allow the proteins to react for 2 hours at room temperature. 6. To quench the reaction, add hydroxylamine to a final concentration of 10mM. This method hydrolyzes nonreacted NHS present on protein #1 and results in hydroxamate. Other quenching methods involve adding 20-50mM Tris, lysine, glycine or ethanolamine; however, these primary amine-containing compounds modify carboxyls on protein #1. 7. Remove excess quenching reagent using a desalting column. Additional Information EDC reacts with a carboxyl group first and forms an amine-reactive O-acylisourea intermediate that quickly reacts with an amino group to form an amide bond and release of an isourea by-product (Figure 1). Primary amine molecule NH Cl NH H2N O 1 OH + O N O 2 N H 1 1 C Carboxylate molecule 2 N N EDC O NH o-Acylisourea reactive ester Amide bond H2N O + H2N Urea Figure 1. One-step EDC reaction with carboxyl and amine-containing molecules. Pierce Biotechnology 3747 N. Meridian Road PO Box 117 Rockford, lL 61105 USA 2 (815) 968-0747 (815) 968-7316 fax Thermofisher.com Information from Our Website • Tech Tip #15: Biotinylate carboxyl groups with EDC and Biotin Hydrazide • Tech Tip #5: Attach an antibody onto glass, silica or quartz surface • Tech Tip #18: Block amino groups to prevent polymer formation in peptide-carrier protein conjugations • Tech Tip #30: Modify and label oligonucleotide 5´ phosphate groups • Tech Tip #3: Determine reactivity of NHS-ester biotinylation and crosslinking reagents • Tech Tip #46: Preferentially biotinylate N-terminal alpha-amino groups in peptides Related Thermo Scientific Products 24500 NHS (N-hydroxysuccinimide), 25g 24510 Sulfo-NHS (N-hydroxysulfosuccinimide), 500mg 24525 Sulfo-NHS, 5g 24520 Sulfo-NHS, No-Weigh™ Format, 8 × 2mg microtubes 26166 Bioconjugate Techniques (Book) 89889 Zeba Spin Desalting Columns, 2mL, 5/pkg 89891 Zeba Spin Desalting Columns, 5mL, 5/pkg 89893 Zeba Spin Desalting Columns, 10mL, 5/pkg 22360 SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate), 50mg 22322 Sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate), 50mg 22622 Sulfo-SMCC, No-Weigh Format, 8 × 2mg microtubes 21555 DSS (disuccinimidyl suberate), 1g 21655 DSS, 50mg 21658 DSS, No-Weigh Format, 8 × 2mg microtubes 21580 BS 3 (bis[sulfosuccinimidyl] suberate), 50mg 21585 BS 3, No-Weigh Format, 8 × 2mg microtubes General References Grabarek, Z. and Gergely, J. (1990). Zero-length crosslinking procedure with the use of active esters. Anal Biochem 185:131-5. Staros, J.V., et al. (1986). Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal Biochem 156:220-2. Timkovich, R. (1977). Detection of the stable addition of carbodiimide to proteins. Anal Biochem 79:135-43. Products are warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“ Documentation”). No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than Buyer. Any model or sample furnished to Buyer is merely illustrative of the general type and quality of goods and does not represent that any Product will conform to such model or sample. NO OTHER WARRANTIES, EXPRESS OR IMPLIED, ARE GRANTED, INCLUDING WITHOUT LIMITATION, IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR ANY PARTICULAR PURPOSE, OR NON INFRINGEMENT. BUYER’S EXCLUSIVE REMEDY FOR NONCONFORMING PRODUCTS DURING THE WARRANTY PERIOD IS LIMITED TO REPAIR, REPLACEMENT OF OR REFUND FOR THE NONCONFORMING PRODUCT(S) AT SELLER’S SOLE OPTION. THERE IS NO OBLIGATION TO REPAIR, REPLACE OR REFUND FOR PRODUCTS AS THE RESULT OF (I) ACCIDENT, DISASTER OR EVENT OF FORCE MAJEURE, (II) MISUSE, FAULT OR NEGLIGENCE OF OR BY BUYER, (III) USE OF THE PRODUCTS IN A MANNER FOR WHICH THEY WERE NOT DESIGNED, OR (IV) IMPROPER STORAGE AND HANDLING OF THE PRODUCTS. Unless otherwise expressly stated on the Product or in the documentation accompanying the Product, the Product is intended for research only and is not to be used for any other purpose, including without limitation, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses, or any type of consumption by or application to humans or animals. Current product instructions are available at thermofisher.com. For a faxed copy, call 800-874-3723 or contact your local distributor. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All (other) trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA. Pierce Biotechnology 3747 N. Meridian Road PO Box 117 Rockford, lL 61105 USA 3 (815) 968-0747 (815) 968-7316 fax Thermofisher.com ">

Public link updated
The public link to your chat has been updated.
Advertisement