實(shí)驗(yàn)指南-SMPH交聯(lián)劑（制備抗體）

SMPH是一種具有N-羥基琥珀酰亞胺（NHS）酯和馬來酰亞胺基團(tuán)，允許胺和巰基分子的共價(jià)結(jié)合的異功能交聯(lián)劑。NHS酯與伯胺在PH7- 9反應(yīng)生成酰胺鍵，而馬來酰亞胺在pH6.5-7.5處與巰基反應(yīng)形成穩(wěn)定的硫醚鍵。在水溶液中，水解NHS酯是一種競(jìng)爭(zhēng)反應(yīng)，其反應(yīng)速率隨pH的增加而增加。馬來酰亞胺基團(tuán)比NHS酯基穩(wěn)定，但在pH值大于7.5時(shí)，對(duì)巰基的水解反應(yīng)也會(huì)失去特異性。由于這些原因，涉及這型的共軛實(shí)驗(yàn)，異官能團(tuán)交聯(lián)劑通常在pH 7.2-7.5下進(jìn)行，NHS酯（胺靶向）反應(yīng)是在馬來酰亞胺（巰基靶）反應(yīng)之前或同時(shí)完成的。

SMPH可用于在兩步反應(yīng)方案中制備抗體和半抗原載體蛋白綴合物。先，含胺的蛋白質(zhì)與交聯(lián)劑的幾倍摩爾過量反應(yīng)，然后通過透析除去過量的（未反應(yīng)的）試劑；后，加入巰基分子以與已經(jīng)附著于*親合物的馬來酰亞胺基團(tuán)反應(yīng)。

SMPH交聯(lián)劑對(duì)水分。在溫度下在干燥劑中儲(chǔ)存試劑瓶。在打開前平衡小瓶室溫，以避免容器內(nèi)的濕氣凝結(jié)。溶解所需量的試劑，并在水解發(fā)生前立即使用。丟棄未使用的重組試劑。

避免在共軛過程中含有初級(jí)胺（例如TrI或甘氨酸）和巰基的緩沖液，因?yàn)樗鼈儠?huì)與預(yù)期的反應(yīng)競(jìng)爭(zhēng)。如果需要，將樣品透析成適當(dāng)?shù)木彌_液，如磷酸鹽緩沖鹽水（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

兩步蛋白質(zhì)交聯(lián)工藝

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準(zhǔn)備材料：

• 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過程:

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.

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產(chǎn)品列表：

Purified Enzymes純化的：

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

BS CAS #: 58626-38-3

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

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

BMPS CAS #: 55750-62-4

EMCS CAS #: 55750-63-5

GMBS CAS #: 80307-12-6

LC-SPDP

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