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junshane木虫 (正式写手)
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[求助]
含氨基官能团的小分子接枝改性聚丙烯酸纳米纤维膜，求助，谢谢！
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| RT，最近制备出了一种聚丙烯酸（PAA）的纳米纤维膜（不溶于水），想在纤维表面用含氨基官能团的小分子加以改性，基本上就是常规的酰胺化反应。常见的方法是先把PAA的羧基酰氯化，再接枝氨基小分子，但是考虑这种方法会对纳米纤维的形态构成很大的影响，所以想寻求一种温和的方法，在以水为反应体系以及不牺牲纳米纤维完整形态的前提下，完成酰胺化反应，望各位虫友相助，谢谢！ |
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Number Description 22980 EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), 5g 22981 EDC, 25g 77149 EDC, 10mg Molecular Weight: 191.7 CAS # 25952-53-8 Storage: Upon receipt store Product No. 22980 and 22981 desiccated at -20°C. Store Product No. 77149 at 4°C. EDC is shipped at ambient temperature. Introduction The 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. 5. React for 2 hours at room temperature. 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. 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). |
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3楼2014-12-04 11:15:17
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