蛋白交联、修饰和标记是研究蛋白结构和相互作用的常用技术。 交联是指将两个或多个分子通过共价键化学结合的过程。 修饰是指连接或裂解化学基团，以改变原始分子的溶解性或其他属性。

 

产品名称	SMPH
英文名称	succinimidyl-6-((b-maleimidopropionamido)hexanoate
单体	N-羟基琥珀酰亚胺，马来酰亚胺基团
分子式	C17H21N3O7
分子量	379.4
溶解度	DMSO or DMF
纯度	98%
保存	-20
结构式	http://s2/small/003ap78czy7jWdV1UVrb1&690

 

 

 

SMPH是一种具有N-羟基琥珀酰亚胺（NHS）酯和马来酰亚胺基团，允许胺和巯基分子的共价结合的异功能交联剂。NHS酯与伯胺在PH7- 9反应生成酰胺键，而马来酰亚胺在pH6.5-7.5处与巯基反应形成稳定的硫醚键。在水溶液中，水解NHS酯是一种竞争反应，其反应速率随pH的增加而增加。马来酰亚胺基团比NHS酯基更稳定，但在pH值大于7.5时，对巯基的水解反应也会失去特异性。由于这些原因，涉及这种类型的共轭实验，异官能团交联剂通常在pH 7.2-7.5下进行，NHS酯（胺靶向）反应是在马来酰亚胺（巯基靶）反应之前或同时完成的。

 

SMPH可用于在两步反应方案中制备抗体酶和半抗原载体蛋白缀合物。首先，含胺的蛋白质与交联剂的几倍摩尔过量反应，然后通过透析除去过量的（未反应的）试剂；最后，加入巯基分子以与已经附着于第一亲合物的马来酰亚胺基团反应。

 

SMPH交联剂对水分敏感。在指定温度下在干燥剂中储存试剂瓶。在打开前平衡小瓶至室温，以避免容器内的湿气凝结。溶解所需量的试剂，并在水解发生前立即使用。丢弃任何未使用的重组试剂。

 

避免在共轭过程中含有初级胺（例如TrI或甘氨酸）和巯基的缓冲液，因为它们会与预期的反应竞争。如果需要，将样品透析成适当的缓冲液，如磷酸盐缓冲盐水（PBS）。

 

Molecules to be reacted with the maleimide moiety must have free (reduced) sulfhydryls. Reduce peptide disulfide bonds with Thermo Scientific Immobilized TCEP Disulfide Reducing Gel (Product No. 77712). Reduce disulfide bonds in high molecular weight proteins using 5mM TCEP (1:100 dilution of Thermo Scientific Bond-Breaker TCEP Solution, Product No. 77720) for 30 minutes at room temperature, followed by two passes through an appropriate desalting column (e.g., Thermo Scientific Zeba Spin Desalting Columns). Be aware that proteins (e.g., antibodies) may be inactivated by complete reduction of disulfide bonds they contain. Selective reduction of hinge-region disulfide bonds in IgG may be accomplished with 2-Mercaptoethylamine•HCl (2-MEA, Product No. 20408). Sulfhydryls may be added to molecules using N-succinimidyl S-acetylthioacetate (SATA, Product No. 26102) or 2-iminothiolane•HCl (Traut’s Reagent, Product No. 26101), which modify primary amines.

 

Procedure for Two-step Protein Crosslinking

两步蛋白质交联工艺

Generally, a 10- to 50-fold molar excess of crosslinker over the amount of amine-containing protein results in sufficient maleimide activation to enable several sulfhydryl-containing proteins to be conjugated to each amine-containing protein.

 

More dilute protein solutions require greater fold molar excess of reagent to achieve the same level of activation. Empirical testing is necessary to determine activation levels and final conjugation ratios that are optimal for the intended application.

 

 

A. Material Preparation准备材料：

• Conjugation Buffer: Phosphate buffered saline (PBS, pH 7.2; e.g., Product No. 28372) or other amine- and sulfhydrylfree buffer at pH 6.5-7.5 (see Important Product Information) – adding EDTA to 1-5mM helps to chelate divalent metals,

thereby preventing disulfide formation in the sulfhydryl-containing protein

• Desalting column to separate modified protein from excess cross-linker and reaction byproducts (e.g., Zeba™ Spin Desalting Columns)

• Amine-containing protein (Protein-NH2) and sulfhydryl-containing protein (Protein-SH) to be conjugated

 

B.Conjugation过程:

Note: For best results, ensure that Protein-SH is prepared (see Important Product Information) and ready to combine with Protein-NH2 in step 5. 

1. Dissolve Protein-NH2 in Conjugation Buffer at 0.1mM (e.g., 5mg in 1mL for a 50kDa protein). 

2. Add crosslinker to dissolved Protein-NH2 at 1mM final (= 10-fold molar excess) by dissolving 3.80mg SMPH in 1mL DMSO (makes 10mM) and then adding 100µL/mL of Protein-NH2 solution.

3. Incubate reaction mixture for 30 minutes at room temperature or 2 hours at 4°C.

4. Remove excess crosslinker using a desalting column equilibrated with Conjugation Buffer.

Note: Follow the desalting column product instructions to determine which fractions contain Protein-NH2. Alternatively, locate the protein by measuring for fractions having peak absorbance at 280nm; however, be aware that the NHS-ester leaving group also absorbs strongly at 280nm.

5. Combine and mix Protein-SH and desalted Protein-NH2 in a molar ratio corresponding to that desired for the final conjugate and consistent with the relative number of sulfhydryl and activated amines that exist on the two proteins.

6. Incubate the reaction mixture at room temperature for 30 minutes or 2 hours at 4°C.

Note: Generally, there is no harm in allowing the reaction to proceed for several hours or overnight, although usually the reaction will be complete in the specified time. To stop the conjugation reaction before completion, add buffer containing reduced cysteine at a concentration several times greater than the sulfhydryls of Protein-SH.

Note: Conjugation efficiency may be estimated by electrophoresis separation and subsequent protein staining.

 

产品名称：SMPB

简介：SMPB crosslinker (N-Succinimidyl 4-[4-maleimidophenyl]butyrate) is a heterobifunctional pro-tein crosslinking reagent that is reactive toward sulfhydryl and amino groups. The SMPB pro-tein crosslinker is an extended chain length analog of MBS crosslinker.

http://s5/mw690/003ap78czy7jWdQql4E44&690

Molecular Weight: 356.3

CAS #: 79886-55-8

Spacer Length: 11.6 Å

Alternative Name: 4-(4-Maleimidophenyl)butyric acid N-hydroxysuccinimide ester

 

产品名称：SMCC

简介：SMCC crosslinker (Succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate) is a heterobi-functional protein crosslinker with a maleimide group that is sulfhydryl (thiol; -SH) reactive and a NHS ester group that is amine reactive. The SMCC crosslinking reagent is permeable across the lipid bilayer of the cell membrane. The 9 atom spacer arm of SMCC is not cleavable.

http://s5/mw690/003ap78czy7jWdLmnyY74&690

Molecular Weight: 334.3

CAS #: 64987-85-5

Spacer Length: 8.3 Å

Alternative Names: 4-(N-Maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester;

 

 

产品列表：

BMPS CAS #: 55750-62-4

EMCS CAS #: 55750-63-5

GMBS CAS #: 80307-12-6

LC-SPDP

MBS CAS #: 58626-38-3

Sulfo-NHS   CAS #: 106627-54-7

BS3 CAS #: 82436-77-9

BS2G

BSG CAS #: 79642-50-5

DSP CAS #: 57757-57-0

DSS CAS #: 68528-80-3

DTSSP CAS #: 81069-02-5

EGS CAS #: 70539-42-3

Sulfo-EGS

DSSeb CAS # 23024-29-5

PDPH

SBA CAS #: 42014-51-7

SIA CAS #: 39028-27-8

SMCC CAS #: 64987-85-5

SMPB CAS #: 79886-55-8

SMPH

SPDP CAS #: 68181-17-9

Sulfo-LC-SPDP

Sulfo-MBS

Sulfo-SANPAH CAS #: 102568-43-4

Sulfo-SMCC CAS #: 92921-24-9

BS2G-d4

BS3-d4

DSG-d4

DSS-d4

DSP-d8

Biotin Hydrazide CAS #: 66640-86-6

NHS-Biotin CAS #: 35013-72-0

NHS-SS-Biotin CAS #: 142439-92-7

Sulfo-NHS-Biotin CAS #: 119616-92-5

Sulfo-NHS-LC-Biotin CAS #: 128062-22-0

Sulfo-NHS-SS-Biotin CAS #: 202057-28-1

HPG CAS #: 24645-80-5

SATA CAS #: 76931-93-6

Sulfo-NHS Acetate CAS #: 152305-87-8

Purified Enzymes纯化的酶：

Pepsin       CAS Number: 9001-75-6

Chymotrypsin CAS Number: 9004-07-3

Trypsin      CAS Number: 9002-07-7

Streptavidin CAS Number: 9013-20-1

Horseradish Peroxidase (HRP)   CAS Number: 9003-99-0

Alkaline Phosphatase 35K Units CAS Number: 9001-78-9

Purified Polysorbate 20

Purified Polysorbate 80

Purified Nonidet P-40

Purified Triton X-100

Purified Triton X-114

TCEP-HCl Reducing Agent   CAS Number: 51805-45-9

DTT    CAS Number: 3483-12-3

pNPP   CAS Number: 4264-83-9

Sodium meta-Periodate  CAS Number: 7790-28-5

Succinylacetone        CAS Number: 51568-18-4

Dimethyl sulfoxide     CAS Number: 67-68-5

Dimethylformamide      CAS Number: 68-12-2

MES Monohydrate        CAS Number: 145224-94-8

Sodium Borate Buffer   CAS Number: 1303-96-4