Protein Crosslinking

Crosslinkers contain reactive ends to specific functional groups, such as primary amines and sulfhydryls on proteins and antibodies, enabling the covalent joining of two or more biomolecules. Homobifunctional crosslinkers have the same reactive chemistry at both ends and heterobifunctional crosslinkers have different chemistries at each end. PEGylated crosslinkers are also available which provide enhanced solubility, increased stability, reduced aggregation, and reduced immunogenicity to proteins.

Use our Crosslinker Selection Tool to find the optimal protein crosslinker reagent for your protein function or interaction application, or access our helpful Bioconjugation Technical Handbook to help improve your crosslinking results.

Crosslinker Selection Tool

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What is protein crosslinking?
Most common crosslinkers
No-weigh packaging crosslinkers
Premium grade crosslinking reagents
In vivo protein crosslinking

What is protein crosslinking?

Crosslinkers, also known as bifunctional crosslinkers, are reagents that contain two or more reactive groups which covalently attach via a spacer to functional groups on proteins or other biomolecules. Three types of crosslinkers are available: homobifunctional crosslinkers, heterobifunctional crosslinkers, and photoreactive crosslinkers. Homobifunctional crosslinking reagents have identical reactive groups, primarily amine-to-amine or sulfhydryl-to-sulfhydryl. They are typically used to form intramolecular crosslinks or to prepare polymers from monomers. Heterobifunctional crosslinking reagents have different reactive groups such as amine-to-sulfhydryl, carboxyl-to-amine, or sulfhydryl-to-carboxyl. They are useful for preparing conjugates between two different biomolecules. Heterobifunctional crosslinking reagents also include photoreactive crosslinking reagents that react with nucleophiles or form C-H insertion sites after exposure to UV light. Learn more about crosslinking reagents.

Find the optimal crosslinking reagent for your application using our Crosslinker Selection Tool or select from the most common crosslinkers below.

Most common crosslinkers based on type and reactive chemistry

Amine-to-amine
Sulfhydryl-to-sulfhydryl

Amine-to-amine

Reactive group	Order products	Spacer Arm (Å)	Cleavable?	Water-soluble?	Membrane-permeable?	Reversible?
NHS ester	DSG	7.7	N/A	No	Yes	No
	DSS	11.4	N/A	No	Yes	No
	BS3	11.4	N/A	Yes	No	No
	BS(PEG)9	35.8	N/A	Yes	No	No
	DSP	12.0	Thiols	No	Yes	No
	DTSSP	12.0	Thiols	Yes	No	No
	EGS	16.1	Hydoxylamine	No	Yes	Yes

Sulfhydryl-to-sulfhydryl

Sulfhydryl -to-Sulfhydryl Crosslinkers	Order product	Spacer Arm (Å)	Cleavable?	Water-soluble?	Membrane permeable?
NHS-Maleimide	SMCC	8.3	N/A	No	Yes
NHS-Maleimide	Sulfo-SMCC	8.3	N/A	Yes	No
NHS-Pyridyldithiol Crosslinkers	SPDP	6.8	Thiol	No	Yes
NHS-Pyridyldithiol Crosslinkers	LC-SPDP	15.7	Thiol	No	Yes

Mechanism of DSG crosslinking simultaneously with two amine molecules

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Homobifunctional crosslinking.DSG, an amine-to-amine crosslinker, contains NHS ester reactive groups at opposite ends of the short-length spacer arm. Homobifunctional crosslinkers allow for simultaneous reactions with two molecules.

Amine-to-sulfhydryl
Carboxyl-to-amine

Amine-to-sulfhydryl

Reactive group	Order product	Spacer Arm (Å)	Cleavable?	Water-soluble?	Membrane permeable?
NHS-Maleimide
	SMCC	8.3	N/A	No	Yes
	Sulfo-SMCC	8.3	N/A	Yes	No
NHS-Pyridyldithiol Crosslinkers	SPDP	6.8	Thiol	No	Yes
NHS-Pyridyldithiol Crosslinkers	Sulfo-LC-SPDP	15.7	Thiol	Yes	No

Carboxyl-to-amine

Reactive group	Order product	Cleavable?	Water-soluble?	Membrane permeable?
Carbodiimide / NHS ester
	EDC	No	Yes	No
	NHS	No	No	Yes
	Sulfo-NHS	No	Yes	No

Mechanism of sulfo-smcc crosslinking with an amine and sulfhydryl molecule

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Heterobifunctional crosslinking.Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), an amine-to-sulfhydryl crosslinker, contains NHS-ester and maleimide reactive groups at opposite ends of a medium-length cyclohexane-stabilized spacer arm. Heterobifunctional crosslinkers allow for sequential reactions with two molecules to control the conjugation without polymerization.

Reactive group	Order product	Spacer Arm (Å)	Cleavable by?	Water-soluble?	Membrane permeable?
NHS ester/ aryl azide
NHS ester/ aryl azide	Sulfo-SANPAH	18.2 Long	No	Yes	No
NHS ester/ diazirine	SDA	3.9 Short	No	No	Yes
	Sulfo-SDA	3.9 Short	No	Yes	No
	LC-SDA	12.5 Mid	No	No	Yes
	Sulfo-LC-SDA	12.5 Mid	No	Yes	No
	SDAD	13.5 Mid	Thiols	No	Yes
	Sulfo-SDAD	13.5 Mid	Thiols	Yes	No

NHS ester and diazirine crosslinker reacting with an amine group then undergoing photoactivation, respectively

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Photoreactive crosslinking. N-hydroxysuccinimide (NHS) esters react efficiently with primary amine groups (NH2) in pH 7-9 buffer to form stable amide bonds upon release of NHS. Photoactivation of diazirine with long-wave UV light (330-370nm) creates reactive carbene intermediates. Such intermediates can form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths.

Heterobifunctional kits

Heterobifunctional kits

Heterobifunctional kits are offered to conjugate and purify a protein with a reactive maleimide or DBCO label for reaction with a sulfhydryl or azide, respectively.

Reactive group	Order product	Spacer Arm (Å)	Labeling scale	Number of reactions	Labeled protein reactive towards
TFP Ester/DBCO	DBCO Protein Labeling Kit	17.9	0.1–0.5 mg	5	Azides
NHS-Maleimide	Maleimide Protein Labeling Kit	8.3	0.1–1 mg	5	Sulfhydryls
NHS-Maleimide	Controlled Protein-Protein Crosslinking Kit	11.6 2.8	0.5-5 mg	1	Amine Sulfhydryl

* Not determined

Homobifunctional

Amine-to-amine
Sulfhydryl-to-sulfhydryl

Amine-to-amine

Reactive group	Order products	Spacer Arm (Å)	Cleavable?	Water-soluble?	Membrane-permeable?	Reversible?
NHS ester	DSG	7.7	N/A	No	Yes	No
	DSS	11.4	N/A	No	Yes	No
	BS3	11.4	N/A	Yes	No	No
	BS(PEG)9	35.8	N/A	Yes	No	No
	DSP	12.0	Thiols	No	Yes	No
	DTSSP	12.0	Thiols	Yes	No	No
	EGS	16.1	Hydoxylamine	No	Yes	Yes

Sulfhydryl-to-sulfhydryl

Sulfhydryl -to-Sulfhydryl Crosslinkers	Order product	Spacer Arm (Å)	Cleavable?	Water-soluble?	Membrane permeable?
NHS-Maleimide	SMCC	8.3	N/A	No	Yes
NHS-Maleimide	Sulfo-SMCC	8.3	N/A	Yes	No
NHS-Pyridyldithiol Crosslinkers	SPDP	6.8	Thiol	No	Yes
NHS-Pyridyldithiol Crosslinkers	LC-SPDP	15.7	Thiol	No	Yes

Click image to enlarge

Heterobifunctional

Amine-to-sulfhydryl
Carboxyl-to-amine

Amine-to-sulfhydryl

Reactive group	Order product	Spacer Arm (Å)	Cleavable?	Water-soluble?	Membrane permeable?
NHS-Maleimide
	SMCC	8.3	N/A	No	Yes
	Sulfo-SMCC	8.3	N/A	Yes	No
NHS-Pyridyldithiol Crosslinkers	SPDP	6.8	Thiol	No	Yes
NHS-Pyridyldithiol Crosslinkers	Sulfo-LC-SPDP	15.7	Thiol	Yes	No

Carboxyl-to-amine

Reactive group	Order product	Cleavable?	Water-soluble?	Membrane permeable?
Carbodiimide / NHS ester
	EDC	No	Yes	No
	NHS	No	No	Yes
	Sulfo-NHS	No	Yes	No

Click image to enlarge

Photoreactive

Reactive group	Order product	Spacer Arm (Å)	Cleavable by?	Water-soluble?	Membrane permeable?
NHS ester/ aryl azide
NHS ester/ aryl azide	Sulfo-SANPAH	18.2 Long	No	Yes	No
NHS ester/ diazirine	SDA	3.9 Short	No	No	Yes
	Sulfo-SDA	3.9 Short	No	Yes	No
	LC-SDA	12.5 Mid	No	No	Yes
	Sulfo-LC-SDA	12.5 Mid	No	Yes	No
	SDAD	13.5 Mid	Thiols	No	Yes
	Sulfo-SDAD	13.5 Mid	Thiols	Yes	No

Click image to enlarge

Heterobifunctional kits

Heterobifunctional kits

Heterobifunctional kits

Heterobifunctional kits are offered to conjugate and purify a protein with a reactive maleimide or DBCO label for reaction with a sulfhydryl or azide, respectively.

Reactive group	Order product	Spacer Arm (Å)	Labeling scale	Number of reactions	Labeled protein reactive towards
TFP Ester/DBCO	DBCO Protein Labeling Kit	17.9	0.1–0.5 mg	5	Azides
NHS-Maleimide	Maleimide Protein Labeling Kit	8.3	0.1–1 mg	5	Sulfhydryls
NHS-Maleimide	Controlled Protein-Protein Crosslinking Kit	11.6 2.8	0.5-5 mg	1	Amine Sulfhydryl

* Not determined

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No-weigh packaging crosslinkers

Protein crosslinking no-weigh packaging formats offer quick and convenient ready-to-use solutions. This format eliminates the need to weigh out small amounts of dry compounds and eliminates the waste of unused material. Simply reconstitute and the reagent is ready to use at the desired concentration.

Premium grade crosslinking reagents

Protein crosslinking premium grade reagents are high-quality reagents formulated for applications where product integrity and risk minimalization are essential. These premium grade reagents are ideal for long-term research projects that require consistent performance. Crosslinker premium grade products provide quality assurance review, lot sample retention, change control notification, along with an extra level of analytical testing and chemical characterization.

Comparison of specifications between premium and standard grade reagents

Test parameter	Test method	Premium grade	Standard grade
Purity	Quantitative NMR using an internal standard	Yes	Yes
Visual	Color assessment	Yes	Yes
Solubility	Sample dissolves at specified concentration in deionized water to yield a clear, colorless solution	Yes	Yes
Identity	Infrared spectroscopy	Yes	Yes
Mass identity	Mass spectrometry	Yes	NA
Water content	Karl Fischer titration	Yes	NA
Trace metals	Inductively coupled plasma mass spectrometry (ICP-MS)	Yes	NA
Elemental analysis	Combustion analysis (values reported for C, H, N, O, and S)	Yes	NA
Residual solvent analysis	Headspace gas chromatography	Upon request	NA

In vivo protein crosslinking

In vivo crosslinking can be used to characterize protein interactions and ligand-receptor interactions irrespective of treatment conditions. Various sizes of in vivo crosslinkers are available to target surface and intracellular proteins for analysis by different methods such as immunoprecipitation (IP), Co-IP, chromatin immunoprecipitation (ChIP), electrophoresis mobility shift assay (EMSA), western blot, immunofluorescence (IF), and immunohistochemistry (IHC).

Homobifunctional, amine-reactive, NHS-ester crosslinkers

NHS-ester crosslinkers are widely used for in vivo crosslinking. NHS-ester, amine-specific reactions are fast and highly efficient since lysine residues are abundant and easily accessible on the surface of most proteins. Homobifunctional crosslinkers use a “shotgun” type approach for identifying protein complexes since they crosslink all interacting molecules whose lysine residues are within the crosslinkers spacer length. Subsequent detection simply requires a specific antibody or probe that targets one of the molecules in the complex.

Find homobifunctional, amine-reactive, NHS-ester crosslinkers for in vivo crosslinking

Cell surface protein crosslinking
- BS3 (= Sulfo-DSS), 11.4 Å spacer (Ref 7)
- DTSSP, 12.0 Å spacer, cleavable
- Sulfo-EGS, 16.1 Å spacer, cleavable
Intracellular protein crosslinking
- DSG, 7.7 Å spacer (Ref 1, 3)
- DSP, 12.0 Å spacer, cleavable (Ref 4, 6)
- DSS, 11.4 Å spacer (Ref 3)
- EGS, 16.1 Å spacer, cleavable (Ref 2, 5)

Chemical structure of homobifunctional crosslinker DSG

Positive STAT3 bands with DSG and DSS, and combined Photo-Leucine and Photo-Methionine crosslinkers compared to negative control

Comparison of several in vivo crosslinking methods. HeLa cells treated with 1% Formaldehyde (HCHO) or 1mM homobifunctional NHS-ester crosslinker (Thermo Scientific DSG and DSS) in PBS for 10 minutes before quenching. A fourth set of HeLa cells were treated and crosslinked for 10 minutes with 4mM Photo-Leucine, 2mM Photo-Methionine (Photo-AA) according to the procedure (see subsequent section). Formaldehyde-treated and NHS-ester-treated cells were quenched with 100mM glycine pH 3 and 500mM Tris pH 8.0, respectively for an additional 15 minutes. One million cells from each condition were then lysed and 10µg of each sample was heated at 65°C for 10 minutes in reducing sample buffer containing 50mM DTT and analyzed by SDS-PAGE and Western blotting with Stat3 specific antibodies (Cell Signaling). Gapdh (Santa Cruz) and beta-actin (US Biologicals) were blotted as loading controls.

Heterobifunctional, photoreactive, NHS-ester/diazirine crosslinkers

NHS-ester/diazirine crosslinkers combine NHS-ester chemistry with diazirine-based photochemistry to create crosslinking. While typical heterobifunctional crosslinkers require specific amino acid groups at correct distances, SDA crosslinkers utilize a “two-step” reaction by crosslinking one protein using the NHS-ester then UV light to activate crosslinking via the diazirine moiety to any amino acid on a second protein.

Find heterobifunctional, photoreactive, NHS-ester/diazirine crosslinkers for in vivo crosslinking

Cell surface protein crosslinking
- Sulfo-SDA, 3.9 Å spacer
- Sulfo-LC-SDA, 12.5 Å spacer
- Sulfo-SDAD, 13.5 Å spacer, cleavable
Intracellular protein crosslinking
- SDA, 3.9 Å spacer
- LC-SDA, 12.5 Å spacer
- SDAD, 13.5 Å spacer, cleavable

Chemical structure of heterobifunctional crosslinker LC-SDA

Positive EEA1 crosslinking using SDA and SDAD (-DTT) compared to Sulfo-SDA, SDAD (+DTT), and control

Intracellular crosslinking of EEA1 protein complex using NHS-ester diazirine crosslinkers. HeLa cells (2 x 10^6) were labeled for 10 minutes with 1mM SDA, Sulfo-SDA or SDAD in PBS. Non-reacted NHS-esters were quenched with Tris•HCl, pH 8.0 at a final concentration of 100mM for 5 minutes and then rinsed with PBS. NHS-diazirine labeled cells and a mock-treated control (-) were UV irradiated using a Stratalinker 2400 at 365nm for 15 minutes at a distance of 4 cm in PBS. After UV treatment, cells were lysed with Thermo Scientific M-PER Mammalian Protein Extraction Reagent and analyzed for total protein concentration using the Thermo Scientific Pierce BCA Protein Assay. Reducing sample buffer was added to 10 µg of each sample except for a duplicate SDAD treated sample (-DTT) and separated by SDS-PAGE. Results were analyzed by Western blot using anti-EEA1 and GAPDH antibodies.

Photoreactive amino acid crosslinkers

Photo-Leucine and Photo-Methionine, used in metabolic labeling, are incorporated into proteins by protein synthesis (3). These non-toxic photo-amine acids are non-invasive and do not alter the cell’s metabolism. Photo-Leucine and Photo-Methionine are highly specific and ideal for studying hydrophobic interactions. Further, they can be used for either extracellular or intracellular crosslinking.

Find photoreactive amino acid crosslinkers for in vivo crosslinking

photoreactive crosslinkers added to cells then exposed to UV light show crosslinking by western blot analysis

Protocol summary for in vivo labeling and crosslinking using photoreactive amino acids as crosslinkers.

positive crosslinking using photoreactive amino acids versus formaldehyde

Photo-reactive amino acid and formaldehyde crosslinking are complementary techniques for protein interaction analysis. HeLa cells were mock treated (Lane 1), treated with 1% formaldehyde for 10 minutes (Lane 2), or treated with Photo-Methionine and Photo-Leucine followed by UV treatment (Lane 3). Cells were lysed and 10µg of each was analyzed by SDS-PAGE and Western blotting with antibodies against HSP90, STAT3, Ku70 and Ku86. Lower panels are beta-actin (upper band) and GAPDH (lower band), which were blotted as loading controls.