INSTRUCTIONS
3747 N. Meridian Road
SMPT P.O. Box 117
Rockford, IL 61105
Sulfo-LC-SMPT
21558 21568 0380.1
Number Description
21558 SMPT (4-succinimidyloxycarbonyl-伪-methyl-伪-[2-pyridyldithio]toluene), 50 mg
Molecular Weight: 388.46 O
N
Spacer Arm: 11.2 脜 NO SS
Formula: C18H16N2O4S2
O
O
Storage: Upon receipt store product desiccated at 4掳C. Product is shipped at ambient temperature.
21568 Sulfo-LC-SMPT (sulfosuccinimidyl 6-[伪-methyl-伪-(2-pyridyldithio)toluamido]hexanoate), 50 mg
Molecular Weight: 603.67 O
O
Na O O
Spacer Arm: 20.0 脜 N N N
S H
O
O S
O
Formula: C24H26N3NaO8S3 S
O
Storage: Upon receipt store product desiccated at -20掳C. Product is shipped at ambient temperature.
Introduction
SMPT is a heterobifunctional sulfhydryl- and amine-reactive cross-linker. SMPT is used often for conjugating a toxin
molecule to a monoclonal antibody directed against a cell-surface antigen. These immunotoxins produced with SMPT are
highly potent as the cleavable disulfide imparts increased cytotoxicity compared to conjugates without a cleavable bond, such
as those produced with SPDP. Also, next to the pyridine-2-thione in the spacer arm are a benzene ring and a methyl group
adjacent to a carbon that hinders the disulfide bond, allowing exceptional conjugate stability in vivo. SMPT has the added
benefit of being more stable to hydrolysis in aqueous solutions than other NHS cross-linkers.
Sulfo-LC-SMPT is a water-soluble version of SMPT with an extended spacer arm (20.0 脜). The extended spacer arm
increases reactivity and minimizes steric hindrance effects, which can occur when conjugating an antibody to a toxin. The
NHS ester on Sulfo-LC-SMPT is not as stable in aqueous solutions as it is in SMPT. Additionally, the disulfide bond in
Sulfo-LC-SMPT is not as stable as it is in SMPT; however, disulfide bonds formed by Sulfo-LC-SMPT with sulfhydryl-
containing molecules are more stable in solution than those formed by SPDP.
Important Product Information
鈥? Dissolve SMPT in an organic solvent, such as acetonitrile, before adding to a buffer. If acetonitrile is incompatible with
the application, DMF and DMSO may be used for solubilization provided it is added immediately to the buffer solution
containing the protein. SMPT is more stable in acetonitrile than in DMF or DMSO.
鈥? Sulfo-LC-SMPT is water-soluble and can be added as a solid directly to the reaction or prepared as a concentrated
solution in the reaction buffer and immediately added to the protein. To prevent condensation in the vial and reagent
hydrolysis, allow vial to equilibrate to room temperature before opening.
鈥? Store SMPT stock solutions prepared in acetonitrile frozen and moisture-free. SMPT dissolved in water and stored at
room temperature for 16 hours will have a 5% reactivity reduction from NHS ester hydrolysis. Reconstituted Sulfo-LC-
SMPT must be used immediately.
鈥? The pyridine-2-thione reacts with free sulfhydryl group(s). Some sulfhydryl-containing molecules oxidize in solution and
form disulfide bonds, which cannot react. The Reduce-Imm鈩? Reducing Kit (Product No. 77700) and Immobilized
TCEP Disulfide Reducing Gel (Product No. 77712) enable disulfide reduction and protein recovery in the absence of
Warranty: Pierce products are warranted to meet stated product specifications and to conform to label descriptions when used and stored properly. Unless otherwise stated, this
warranty is limited to one year from date of sale for products used, handled and stored according to Pierce instructions. Pierce鈥檚 sole liability for the product is limited to
replacement of the product or refund of the purchase price. Pierce products are supplied for laboratory or manufacturing applications only. They are not intended for medicinal,
diagnostic or therapeutic use. Pierce products may not be resold, modified for resale or used to manufacture commercial products without prior written approval from Pierce
Biotechnology. Pierce strives for 100% customer satisfaction. If you are not satisfied with the performance of a Pierce product, please contact Pierce or your local distributor.
reducing agents. Alternatively, sulfhydryls can be introduced via amine modification using N-succinimidyl S-
acetylthioacetate (SATA, Product No. 26102) or 2-iminothiolane鈥Cl (Traut鈥檚 Reagent, Product No. 26101).
General Cross-linking Procedure
Conjugation efficiency is dependent on protein modification level, buffer composition and pH, protein concentration and
reaction time. Modification level depends on the molar ratio of protein to the cross-linker used in the reaction. Increasing the
molar ratio also increases incorporation level. Over-modification of a protein can cause precipitation and activity loss.
Optimal reaction conditions to use must be determined empirically.
A. Materials Required
鈥? Acetonitrile (Product No. 51101)
鈥? Phosphate Buffered Saline (PBS): 0.1 M phosphate, 0.15 M sodium chloride; pH 7.2, (BupH鈩? Phosphate Buffered
Saline Packs, Product No. 28372) or other non-amine-containing buffer at pH 6.5-7.5
鈥? PBS-EDTA: PBS containing 10 mM EDTA
鈥? Amine-containing protein prepared in PBS
鈥? Sulfhydryl-containing protein prepared in PBS-EDTA
鈥? Desalting columns, such as Zeba鈩? Desalt Spin Columns, 2 ml (Product No. 89889), or dialysis units, such as the Slide-
A-Lyzer庐 Dialysis Cassette (Product No. 66380; 10K MWCO, 0.5-3 ml)
B. NHS-ester Reaction
1. Allow cross-linker vial to completely equilibrate to room temperature before opening. Dissolve SMPT in acetonitrile or
Sulfo-LC-SMPT in PBS.
2. Add a volume of cross-linker to the protein to achieve a 2- to 4-fold molar excess of the reagent over the protein. To
minimize damage to the protein, do not exceed 10% solvent in the final reaction mixture. For optimal results, use < 5%
solvent in the reaction.
Note: Typical final concentrations of cross-linker to use are 0.1-10 mM. It may be difficult to keep the conjugate in
solution at cross-linker concentrations above 5 mM.
3. Allow the reaction to proceed for 1-2 hours at room temperature or overnight at 4掳C. If desired, quench the reaction by
adding a solution containing primary amines such as Tris鈥Cl, pH 8.0.
4. Dialyze sample overnight against PBS-EDTA or use a desalting column equilibrated with PBS-EDTA to remove excess
reagent and to exchange the buffer.
C. Pyridine-2-thione Reaction
1. Add the modified protein from Section B to the sulfhydryl-containing protein prepared in PBS-EDTA. React for 12-72
hours at room temperature or 4掳C.
2. If desired, quantify the pyridine-2-thione reaction using a spectrophotometer. The pyridine-2-thione leaving group has an
absorption maximum at 343 nm with an extinction coefficient of 8.08 卤 0.3 脳 103 M-1 cm-1. Quantify conjugation before
quenching the reaction with cysteine (Step 3).
3. To inactivate nonreacted pyridine-2-thione groups, add a final concentration of 0.2 mM cysteine. This treatment will not
affect the disulfide bond in the conjugate.
4. To isolate conjugate, use size exclusion chromatography or ion exchange chromatography.
Information Available on the Web
Please visit the Pierce web site for additional information relating to this product including the following items:
鈥? Tech Tip: Protein stability and storage
鈥? Tech Tip: Determine reactivity of NHS ester biotinylation and cross-linking reagents
鈥? Tech Tip Protocol: Attach an antibody onto glass, silica or quartz surface
In the USA call: 800-8-PIERCE (800-874-3723) or 815-968-0747 鈥? Fax: 815-968-7316 or 800-842-5007 鈥? www.piercenet.com
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B-PER庐 Bacterial Protein Extraction Reagent (in phosphate buffer), 500 ml
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Product References
Na, D. H., et al. (1999). Quantitative analysis of derivatized proteins prepared with pyridyl disulfide-containing cross-linkers by high-performance liquid
chromatography. Bioconjugate Chemistry 10:306-10.
van Oosterhout, Y.V. J. M., et al. (2000). A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host
disease. Blood 95(12):3693-701.
Warren, H.S., et al. (2003). Protective efficacy of CAP18106鈥?138 鈥搃mmunoglobulin G in sepsis. J. Infec. Dis. 188:1382-93.
General References
Blakely, D., et al. (1987). Effect of chemical deglycosylation of Ricin A chain on the in vivo fate and cytotoxic activity of an immunotoxin composed of
ricin A chain and anti-thy 1.1 antibody. Cancer Res. 47:947-52.
Cawley, D., et al. (1978). Homology between ricin and Ricinus communis agglutinin: amino terminal sequence analysis and protein synthesis inhibition
studies. Arch. Biochem. Biophys. 190(2):744-55.
Masuho, Y., et al. (1982). Importance of the antigen-binding valency and the nature of the cross-linking bond in ricin A-chain conjugates with antibody. J.
Biochem. 91:1583-91.
Scott, C., et al. (1987). An immunotoxin composed of a monoclonal antitrasferrin receptor antibody linked by a disulfide bond to the ribsomal inactivating
protein gelonin: potent in vitro and in vivo effects against human tumors. JNCI 79(5):11163-72.
Stuchbury, T., et al. (1975). Reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe
containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. Biochem. J. 151:417-32.
Thorpe, P., et al. (1987). Comparison of two anti-Thy 1.1-abrin A-chain immunotoxins prepared with different cross-linking agents: anti-tumor effects, in
vivo fate, and tumor cell mutants. JNCI 79(5):1101-11.
Sulfo-NHS Technology is protected by U.S. Patent #s 6,407,263, 5,872,261, 5,892,057 and 5,942,628.
Slide-A-Lyzer庐 Dialysis Cassette Technology is protected by U.S. Patent # 5,503,741 and other patent pending.
BCA鈩? Technology is protected by U.S. Patent # 4,839,295.
Current versions of all product instructions are available at piercenet.com. For a faxed copy, call 800-874-3723 or contact your local distributor.
漏Pierce Biotechnology, Inc., 3/2004. Printed in the USA.
In the USA call: 800-8-PIERCE (800-874-3723) or 815-968-0747 鈥? Fax: 815-968-7316 or 800-842-5007 鈥? www.piercenet.com
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