Thermo Scientific™ Pierce™ DSP, No-Weigh™ Format

Catalog No.	Quantity
PI22585	1 g
PIA35393	10 x 1 mg
PI22586	50 mg

Catalog No. PI22585 Supplier Thermo Scientific™ Supplier No. 22585

Description

Crosslink amines to amines with dithiobis succinimidyl propionate, composed of NHS-ester groups and disulfide-containing, 12.0A spacer arm and cleavable with reducing agents.

Thermo Scientific™ Pierce DSP Crosslinker (Lomant's Reagent) is an amine-reactive crosslinker with NHS-ester reactive ends and a cleavable disulfide bond in its spacer arm.

Highlights:

• Cleavable by reducing agents
• Membrane-permeable
• Acylate in aqueous or organic media within two minutes at room temperature
• Four- to five-hour half-life of ester at pH 7.0
• Reactive groups: NHS ester (homobifunctional)
• Reactive toward: amino groups

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Specifications

• Chemical Reactivity: Amine-Amine
• Cleavable: By Thiols
• Description: DSP
• Molecular Weight (g/mol): 404.42
• PEGylated: No
• Spacer Arm Length: 12.0 Å
• Content And Storage: Upon receipt store desiccated at 4°C.
• Cell Permeability: Yes
• Shipping Condition: Ambient
• Product Line: Pierce
• Labeling Method: Chemical Labeling
• Crosslinker Type: Homobifunctional
• Reactive Moiety: NHS Ester
• Spacer: Medium (10 to 30 Å)
• Form: Powder
• Quantity: 1 g
• Solubility: DMF, DMSO
• Format: Standard, Single-use, Premium-grade
• Water Soluble: No
• Product Type: Crosslinker

Frequently Asked Questions (FAQs)

Can you provide the shelf-life for DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent?

DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended. Please see section 8.1 of our Terms & Conditions of Sale (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf) for more details.

Is DSP soluble in acetonitrile?

DSP has been estimated to be soluble in acetonitrile up to a concentration around 25 mg/mL.

Is a hazy solution common after the addition of the stock DSP solution to an aqueous buffer system?

Yes, frequently the resultant reaction mixture will become hazy after the addition of the stock solution.

What are some common uses for the DSP cross-linker?

DSP has been used for, but is not limited to, the following applications: examining spatial relationships of capsid polypeptides, studying renal Na+ and K+ ATPase, nearest neighbor relationships of bovine mitochondrial H+ATPase, chemical cross-linking of a-CPI, identifying cross-linked cytochrome P-450 in rat liver microsomes, studying the influence of metal ions on prothrombin self-association, studying the glycoprotein topology on intact human red blood cells, and determining membrane antigens by covalent cross-linking to monoclonal antibodies.

What is the difference between a homo- and a hetero- bifunctional cross-linker?

A homobifunctional cross-linker has the same reactive group on both ends, for example two amine-reactive groups. It is used in a one-step conjugation where the two molecules are mixed prior to the addition of the cross-linker. This leads to less efficient cross-linking because polymers can easily be created. A heterobifunctional cross-linker has two different reactive groups, for example an amine-reactive group on one end and a sulhydryl reactive group on the other. These can be used in two-step conjugations by reacting the fIrst end with the molecule containing its reactive group, and then adding the molecule that is reactive towards the other end. This leads to much more efficient cross-linking because polymerization does not occur.

How does DSP work as a crosslinker?

DSP is a homobifunctional, water soluble, cleavable, amine-reactive cross-linker. The amine-reactive ends are NHS-esters. This group reacts with primary amines, in proteins that translates to lysine groups and N-terminus amines. It reacts best in the pH range of 7.5 to 8.5, this ensures the amines are deprotonated and it limits the hydrolysis of the ester at higher pHs. NHS-esters are best used in a heavy molar excess over the number of amines present. This helps to overcome the competing hydrolysis of the ester to an inactive form.

How is the DSP cross-linking reaction quenched?

1M Tris (or glycine), pH 7.5 can be added to the reaction mixture to a final concentration of approximately 20-50 mM.

How is DSP cleaved?

The disulfIde bond in DSP's 12Å spacer arm can be cleaved by at least three different methods. The first is by incubating the crosslinker in 10-50 mM DTT for 30 minutes at 37 degrees C. Secondly, the disulfIde can be reduced by incubation in 5% 2-Mercaptoethanol (2-ME) in SDS-PAGE sample buffer at 100 degrees C for 5 minutes. Finally, the “-S-S-” bond can be cleaved by incubation in 100 mM sodium borohydride at room temperature for 30-60 minutes. Other reducing agents such as TCEP-HCl or 2-Mercaptoethylamine (2-MEA) should also work, however these applications have not been referenced.

Can stock solutions of DSP (Lomant's Reagent) be made?

Unfortunately not, the NHS-esters are unstable in aqueous solutions. They will begin to hydrolyze immediately upon contact with water. They cannot be kept in DMSO because it is hygroscopic and will draw moisture out of the air. NHS-crosslinking reagents work best in the pH range of 7.5 to 8.5. Within this range, the half-life of hydrolysis varies from I hour to 15 minutes, so NHS-crosslinking reagents work best when made up fresh.

Will the DSP (Lomant's Reagent) cross-linker penetrate a cell membrane?

Yes, it will penetrate a cell membrane.

What does "store under nitrogen" mean for DSP (Lomant's Reagent)?

A gentle stream of nitrogen gas is held over the opening of the vial to flush the oxygen from the vial. The cap is then immediately placed on the vial. If storing under nitrogen, caution should be taken to ensure that the stream of nitrogen is gentle enough, so that the powdered product will not be blown out of the bottle. If nitrogen is not available, weigh the material as quickly as possible and immediately cap. Store desiccated at 4 degrees C.

What are the recommended storage conditions for DSP (Lomant's Reagent)?

The product is shipped ambient. Upon receipt. the product should be stored at 4 degrees C in a dessicator or with a desiccant bag, and the vial should be warmed to room temperature before opening.

What is the expiration date for DSP (Lomant's Reagent)?

This product has no set expiration date. LSG/Rockford, IL had monitored the bulk material in such a manner that allows us to warrant the product for one year from the date of shipment. This product may still be usable beyond the one year if handled properly. However, using material older than one year is typically not recommended since the material easily hydrolyzes upon exposure to moisture.

How much DSP (Lomant's Reagent) should be used for cross-linking?

A 10 fold molar excess of cross-linker to protein is recommended for protein solutions greater than 5 mg/mL. A 20-50 fold molar excess of cross-linker to protein is recommended for protein solutions less than 5 mg/mL.

Can the DSP cross-linking agent be added directly to the reaction mixture?

No. It must first be dissolved in dry DMSO or DMF.

What is the recommended concentration of the DSP (Lomant's Reagent) stock solution?

An initial stock concentration of 5-10 mM (~3-6 mg/mL) is recommended.

Which components are not recommended in buffers to be used with DSP?

Buffers containing primary amines, Tris, ethanolamine, glycine, or lysine should not be used, since they will quench the reaction. Imidazole and gluathione are also not recommended as buffer components.

What is the recommended sample buffer and reconstitution buffer to obtain optimal cross-linking using DSP (Lomant's Reagent)?

DSP is first dissolved in an organic solvent (such as dry DMSO) and added to an aqueous reaction mixture such as 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5 (PBS). HEPES, bicarbonate/carbonate, and borate buffers may be substituted for PBS. Other buffers may also be substituted provided they do not contain primary amines. Recommended pH is 7-8. Once the cross-linker is reconstituted, use immediately.

Is DSP (Lomant's Reagent) water soluble?

No, the reagent must first be reconstituted utilizing an organic solvent, such as dry DMSO or DMF.
Note: It is recommended that the DMSO/organic solvent should not exceed 10% of the final reaction volume.

Safety and Handling

WARNING: Cancer - www.P65Warnings.ca.gov

For Research Use Only. Not for use in diagnostic procedures.