SMCC-結(jié)構(gòu)反應(yīng)解釋

1、SMCO一類含有N-羥基琥珀酰亞胺（NHS）活性酯和馬來酰亞胺的雙功能偶 聯(lián)劑.可以將分別含有琉基和氨基的化合物鍵接在一起。NHS活性酯與伯胺在PH7-9的環(huán)境形成酰胺鍵。馬來酰胺與琉基在的環(huán)境下形成穩(wěn)定的硫醴鍵。在水 溶液中，NHS舌性酯的水解是（與氨基的反應(yīng)）個(gè)競(jìng)爭(zhēng)反應(yīng)。馬來酰胺比NHS急定，但是在PH大于時(shí)，馬來酰胺會(huì)慢慢水解，失去與琉基反應(yīng)的特異性。因而， 在使用SMCC通常是在的環(huán)境下進(jìn)行，并且先讓 NHS®生反應(yīng)。SMCC結(jié)構(gòu)里 的環(huán)己烷環(huán)可以降低馬來酰胺的水解速率。這使得蛋白質(zhì)在用SMCO飾之后可以凍干存放一段時(shí)間。很多蛋白質(zhì)都選用該試劑來進(jìn)行馬來酰亞胺修 飾。用SMC

2、區(qū)制備抗體-酶或者半抗原作載體的蛋白質(zhì)，經(jīng)常采用兩步合成法。 首先，含有氨基的蛋白質(zhì)與幾倍的偶聯(lián)劑反應(yīng)， 反應(yīng)結(jié)束后通過脫鹽柱或者透析 的方法除掉沒有反應(yīng)玩的 SMCC然后，再與含有琉基的蛋白質(zhì)反應(yīng)。在實(shí)際操 作中要注意的是，SMCCJ白潮濕，存放時(shí)要和干燥劑一起存放。并且使用中從冰 箱拿出來時(shí)要先在室外放置一段時(shí)間平衡溫度，以免立刻開啟，空氣中水分遇冷凝結(jié)，破壞SMCC吉構(gòu)。INSTRUCTIONSSMCC (succinimidyl 4-N-maleimidomethylcyclohexane-1-carboxylate), 50 mgMolecular Weight:Spacer Arm

3、: ? Net Mass Added:Storage: Upon receipt store desiccated at 4°C.Product is shipped at ambient temperature.Sulfo-SMCC (sulfosuccinimidyl 4-N-maleimidomethylcyclohexan e-1-carboxylate), 1 g Sulfo-SMCC, 50 mgSulfo-SMCC, No-Weigh? Format, 8 x 2 mg microtubesMolecular Weight:Spacer Arm: ?Net Mass A

4、dded: CAS #:92921-24-9Storage: Upon receipt store desiccated at -20° C. Produc t is shipped at ambient temperature.IntroductionSMCC and its water-soluble analog Sulfo-SMCC are heterobif unctional crosslinkers that contain N-hydroxysuccinimide (NHS) ester and maleimide groups that allow covalent

5、 conjugation of amine- and sulfhydryl-containingmolecules. NHS estersreactwith primary amines at pH 7-9 to form amide bonds,whilemaleimides react with sulfhydryl groups at pH to form st able thioether bonds. In aqueous solutions, NHS ester hydroly tic degradation is a competing reaction whose rate i

6、ncreases withpH. The maleimide group is more stable than the NHS-estergroup but will slowly hydrolyze and loses its reactionspecificity for sulfhydryls at pH values > . For these re asons, conjugations with these crosslinkers are usually perforreacted beformed at pH with the NHS-ester (amine-targ

7、eted) e or simultaneous with the maleimide (sulfhydryl-targeted) rea ction.The cyclohexane ring in the spacer arm of these reagents decreases therate ofhydrolysisof the maleimide group compared to similar reagents that do not contain this This feature enablesproteinsthat have been maleimide-activate

8、dwith SMCC or Sulfo-SMCC to be lyophilized and stored for late r conjugation to a sulfhydryl-containing molecule. Many maleim ide-activatedprotein products areproducedin this manner (see Related Products).SMCC andSulfo-SMCC areoftenused toprepareantibody-enzyme and hapten-carrier protein conjugates

9、in a two-step reac tion scheme. First, the amine-containing protein is reacted w ith a several-fold molar excess of the crosslinker, followed by removal of excess (nonreacted) reagent by desalting or dialysis; finally, the sulfhydryl-containing molecule is added to react with the maleimide groups al

10、ready attached to the first protein.Sulfo-SMCC is soluble in water and many other aqueous bu ffers toapproximately 10 mM, althoughsolubility decreaseswith increasing salt concentration. SMCC is not directly water -solubleand must bedissolved in anorganicsolvent suchas dimethylsulfoxide (DMSO) or dim

11、ethylformamide (DMF); subseque nt dilution into aqueous reaction buffer is generally possibl e, and most protein reactants will remain soluble if the fi nal concentration of organic solvent is less than 10%.SMCC and Sulfo-SMCCImportant Product Information ?SMCC and Sulfo-SMCC aremoisture-sensitive.S

12、torereagentvial in desiccant.Equilibrate vialto room temperature before opening to avoid moisture condensation inside the container . Dissolve needed amount ofreagent and use it immediatelybefore hydrolysis occurs. Discard any unused reconstituted rea gent. Do not store reagent insolution.?No-Weigh

13、Microtube Handling:Immediatelybeforeuse,puncture the microtube foil with a pipette tip, add 200 仙 l of 50 mM sodium phosphate buffer(pH orultrapurewaterandpipette up and down to mix.After use,cut theusedmicrotube fromthemicrotubestripand discard.Store the unusedmicrotubesinthe foilpouchprovided.Note

14、: Do not use phosphate-buffered saline (PBS) for init ial dissolution of Sulfo-SMCC; the reagent does not dissolve well in buffers exceeding 50 mM total salts. However, once dissolved, the solution can be further diluted in PBS or other non-amine buffers. ?Avoidbuffers containing primaryamines .,Tri

15、s or glycine) and sulfhydryls during conjugation, because they will comp ete withthe intended reaction. Ifnecessary,dialyzeor desalt samples into an appropriate buffer such as phosphate- bu ffered saline (PBS).Molecules to be reacted with the maleimide moiety must h ave free (reduced) sulfhydryls. R

16、educe peptide disulfide bonds with Immobilized TCEP Disulfide Reducing Gel (Product No. 7 7712). For proteins, reduce disulfide bonds using 5 mM TCEP (1:100 dilution of Bond-Breaker ? TCEP Solution, Product No. 77720) for 30 minutes at room temperature,followed by two passes through a suitable desal

17、ting colum n ., Zeba? Desalt Spin Columns). Be aware that proteins ., antibodies) may be inactivated by complete reduction of the irdisulfide bonds. Selective reduction of hinge-region disulfidebonds in IgG can be accomplishedwith2-Mercaptoethylamine? HCl (2-MEA, Product No. 20408). Sulfhydryls can

18、be added to molecules using N-succinimidyl S-acetylthioacetate (SATA, Pr oductNo. 26102) or 2-iminothiolane ? HCl (Traut sReagent, ProductNo. 26101),which modify primary amines.Procedure for Two-step Protein CrosslinkingGenerally, a 10- to 50-fold molar excess of crosslinker over the amount of amine

19、-containing protein results in suffic ient maleimide activation to enable several sulfhydryl-containi ng proteins to be conjugated to each amine-containing protein . More dilute protein solutions require greater fold molar e xcess of reagent to achieve the same activation level. Empir ical testing i

20、s necessary to determine optimal activation lev els and finalconjugationratiosfor the intended application. A. MaterialPreparation?100 mConjugation Buffer: phosphate-buffered saline (PBSM sodium phosphate, 150 mM sodium chloride, pH ;., ProductNo. 28372) or other amine- and sulfhydryl-free buffer at

21、 pH (see Important Product Information) - adding EDTA to 15 mM helps to chelate divalent metals, thereby reducing disu lfide formation in the sulfhydryl-containing protein? Desalting column to separate modified protein from exc ess crosslinker and reaction byproducts ., Zeba Desalt Spin Columns)?Ami

22、ne-containing (Protein-NH2) and sulfhydryl-containing prot eins (Protein-SH) to be conjugatedB. ProtocolNote: For best results, ensure that Protein-SH is prepare d and ready to combine with Protein-NH2 in step 5.1. Prepare Protein-NH2 in Conjugation Buffer.2. Add the appropriate amount of crosslinke

23、r to the prot ein solution. The concentration of the Protein-NH2 determines thecrosslinker molar excess to use. Suggested crosslinker mol ar excesses are as follows(alsosee Table 1):? Protein samples < 1 mg/ml use 40-80-foldmolar excess.?Proteinsamplesof1-4mg/ml use 20-foldmolarexcess.?Proteinsam

24、plesof5-10 mg/ml use 5- to10-fold mola r excess.Table 1. Crosslinker preparation and molar excess to usefor1 ml of sample.Immediately before use, dissolve crosslinker in the appropriate solvent at the concentration denote d inparentheses; then add the listed volume to a 1 ml protein sample. For exam

25、ple, to use the No-Weigh Sulfo-SMCC, d issolve the 2 mg contents of themicrotubein 200仙 lofbufferandthen add theprescribedvolume to per 1mlsample. For the other products, the appropriate amount of dry r eagent must be weighed on a balance ., mg Sulfo-SMCC fordissolution in 500 仙 l buffer).Protein-NH

26、2 Concentration(based on a 50 kDa protein) 10 mg/ml 1 mg/ml mg /mlCrosslinker Molar Excess 5X 20X 50X Sulfo-SMCC(in50mM sodiumphosphateorwater)100 川 mg/ml*)40 ii l mg/ml*) 50 " l mg/ml*) No-Weigh Sulfo-SMCC(in50mM sodiumphosphateorwater)50 屋(10 mg/ml*) 20 膜 (10 mg/ml*) 25 膜 (10 m g/ml*) SMCC(in

27、DMSO or DMF)100iilmg/ml*)100iilmg/ml*)100iilmg/ml*)*Concentration of each crosslinker before adding to protei n sample.Note: If the Sulfo-SMCC solution does not completely dissolve, place the tube under hot running water or incubate fo r several minutes in a 50°Cwater bath.3. Incubate reaction

28、mixturefor 30 minutesat room temperature or 2 hours at4° C.4. Remove excess crosslinkerusing a desalting column equilibrated with Conjugation Buffer.5. Combineand mix Protein-SH anddesaltedProtein-NH2ina molar ratio corresponding tothatdesiredfor the finalconjugate and consistent with the relat

29、ive number of sulf hydryl and activated amines that exist on the two proteins.6. Incubate the reaction mixture at room temperature for 3 0 minutes or 2 hours at4° C.Note: Generally, there is no harm in allowing the reacti on to proceed for several hours or overnight, although usual ly the react

30、ion 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: Conjugatio n efficiency can be estimated by electrophoresis separation a nd subseq

31、uent protein staining.Additional InformationA. Please visit the Pierce website for additional informa tion including the following item: ?Tech Tip: Attach an antibody onto glass, silica or quart z surface B. Two-step reaction schemeMaleimide-activated AntibodyAntibody-enzyme ConjugateSulfo-SMCCAntib

32、odyAntibodyAntibodyEnzymeEnzymeFigure 1. Two-stepreaction scheme forconjugating antibody and enzyme proteinswith Sulfo-SMCC. In this example, thecrosslinker is first reacted with the antibody to produce a maleimide-activated protein. After excess non-reacted crossli nker and by-products are removed,

33、 the maleimide-activated anti body is reacted with the appropriate molar ratio of enzyme having sulfhydryl groups. Usually, several or multiple maleimi de-activations occur per antibodymolecule,enabling severalenzyme molecules to be conjugatedto each antibodymolecule.MBS/BDB/SMCC/sulfo-SMCC1、 SMCC琥珀

34、酰亞胺-4-（N- 馬來酰亞胺）環(huán)已烷 -1-1 羥酸酯分子一端的NHSS旨基團(tuán)與某一蛋白質(zhì)分子的伯氨反應(yīng)形成穩(wěn)定的酰胺鍵， 另 一端（馬來酰亞胺基團(tuán)一端）可與另一蛋白質(zhì)分子的琉基 交聯(lián)。NHS舌性酯與伯 胺在 PH7-9 的環(huán)境形成酰胺鍵。馬來酰亞胺與巰基在的環(huán)境下形成穩(wěn)定的硫醚鍵。在水溶液中，NHS舌性酯的水解是（與氨基的反應(yīng)）個(gè)競(jìng)爭(zhēng)反應(yīng)。馬來酰亞 胺比NHSSl定，1是在PH大于時(shí)，馬來酰亞胺會(huì)慢慢水解，失去與琉基反應(yīng)的 特異性。因而，在使用SMCC通常是在的環(huán)境下進(jìn)行，并且先讓NH弦生反應(yīng)。 SMCC吉構(gòu)里的環(huán)己烷環(huán)可以降低馬來酰亞胺的水解速率。這使得蛋白質(zhì)在用 SMCC窗飾之后可以凍干