Biotinylation

Biochemistry of Proteins

Biotinylation

General Information
Selection guide to protein biotin tools
- Biotinylation of amines
  NHS-Biotin - SulfoNHS-Biotin - NHS-Ic-Biotin - SulfoNHS-Ic-Biotin - SulfoNHS-Ic-Ic-Biotin - NHS-imino-Biotin - SulfoNHS-SS-Biotin -
  Related Products: MTSE-Biotin - Buffers - Desalting Products
  Fluorescent Labeling: FITC
- Biotinylation of thiols
  Maleimido-Biotin - Biocytin-lc-maleimide - BMCC-Biotin - Maleimide-PEO3-Biotin - HPDP-Biotin - Iodoacetyl-lc-Biotin -
- Biotinylation of Aldehydes and Carboxyls (Carbohydrates Nucleic acids)
  Hydrazide-Biotin - Hydrazide-lc-Biotin - AMCH-Biotin - Hydrazide-PEO4-Biotin - Psoralen-PEO4-Biotin - BAPA
- Other biotins
  Biotin - IminoBiotin - Biotin-PEO3-Amine - Biotin-PEO6-Biotin - Biotin-PEO4-Amine - Biotin-PEO12-Biotin - Biotin-Amino-Pentylamine
Main biochemical features of the labeling agents

General information

Labeling reagents are used to tag or modify peptides, proteins (especially antibodies, lectins...) and other molecules carrying similar chemical groups (for example COOH and NH2 found in some glycosylated lipids), purified or present in mixtures, on cells or outside, or lastly immobilized onto gels or surfaces.

Biotin, a very popular and convenient label, very sensitive and versatile

Biotin is very easy to attach to most biomolecules, and then to detect. This 244Da vitamin binds with one of the highest affinity amongst biomolecules, to both avidin (Ka=10-15 M-1) and streptavidin (Ka=10-14 M-1).

This is taken to advantage in detection and separation techniques :

The tremendous affinity of biotin allows to reach sensitivities near to radioactivity in some detection systems! This is a convenient tool:
- to immobilize molecules for the purification or detection (indirect coating of biotinylated Ab).
- to detect a specific probe conjugated to biotin or avidin, and for amplification systems (avidin/biotin complexes labeled by enzymes or fluorophores).

The versatility of biotin/(strept)avidin systems is well appreciated.

The very strong association allows to purify biotinylated molecules, cells or particules, to immobilized-(strept)avidin or -biotin supports. The elution of bound molecules must be performed often with stringent agents like 4M urea (UP031903) or 6M Guanidine (UP018380).

Related Reagents:

Immobilized Avidins and Biotins

Related Reagents:

Labeled Avidins

Selection guide to protein biotin tools

Cat.#	Product		Target-1-	Spacer-2-	Cleav.-3-	Mb perm.-4-	Applications
UP39044	NHS-Biotin	TS	Amines	13.5	no	no	proteins, peptides
UP52117	sNHS-Biotin	TS	Amines	13.5	no	yes	-
UP85262	NHS-lc-Biotin	TS	Amines	22.4	no	no	--
UP54398	sNHS-lc-Biotin	TS	Amines	22.4	no	yes	-
UP29847	sNHS-lc-lc-Biotin	TS	Amines	30.5	no	no	-
UP53031	sNHS-SS-Biotin	TS	Amines	24.3	yes	yes	-
UP35329	NHS-imino-Biotin	-	Amines	-	no	yes	-
UP48198	Maleimido-Biotin	TS	Thiols	--	no	no	proteins, peptides
UP99687	Maleimido-lc-Biocytin	TS	Thiols	-	no	yes	-
UP87284	Maleimide-PEO3-Biotin	TS	Thiols	29.1	no	yes	-
UP55533	Iodoacetyl-lc-Biotin	TS	Thiols	27.1	no	yes	-
UP83035	HPDP-Biotin	TS	Thiols	29.1	yes	yes	-
UP27443	BMCC-Biotin	-	Thiols	32.6	no	yes	-
UP36466	Hydrazide-Biotin	TS	Carbohydrate	4 at.	no	yes	Carbohydrates
UP78631	Hydrazide-lc-Biotin	TS	Carbohydrate	12 at.	no	yes	-
UP84961	BAPA ( and other biotins)	TS	Carboxyls	5 at.	no	yes	DNA, RNA
UPR0756	AMCH-biotin	TS	Aldehyde	-	no	yes	DNA classic sites
UPL7784	Psoralen PEO4-biotin	TS	Thymine/pyrimidine	-	no	yes	nucleic acids, proteins

Main biochemical features of the labeling agents

-1-Targeted chemical group

Main available chemical groups present in biomolecules are amines and carboxyls. Reactive amines (NH2) are often targetted on proteins. Sulfhydryls (SH) or aldehydes (CHO) are present at defined sites or introduced (see SATA reagent) into proteins, peptides, glycoproteins, nucleic acids or lipids.

Once you have choosen the right target, seek the best chemical reactivity for your application.

-2- Spacer arm

The spacer arm separates the reactive group from the label.

The length of the arm should be considered for the stereoscopic availability of the label. Longer spacer is thought to improve the sensitivity of detection, or capture, especially with the big streptavidin molecule (65kDa), and with biotinylated molecules that are immobilized, or present on cells.

The nature of the spacer should be to considered for other various purposes (flexibility, constrained form, iodinatable or cleavable site).

-3- Cleavability

Attaching a label to a biomolecule is useful, but some applications require the label to be removed. An efficient way consists to cleave the spacer arm under controlled conditions. Dissulfide link provides a convenient cleavable spacers, because it can be broken by reducing agents like DTT (UP28425) or TCEP (UP24221).

-4- Membrane permeability / water solubility

The labeling reagents have different hydrophobic patterns, that may affect biological applications. For example, lipophilic reagents cross the biological membranes and label both outside and inside biomolecules of cells. At the opposite, polar reagents should be used if only the outside exposed proteins should be labeled on cells.

Uptima provides for that purpose high quality sulfo derivatives. An additional advantage is that sulfonated reagents can be added directly in aqueous buffer, eliminating the need of organic solvent that may be undesired or toxic (DMSO).

-5- Detection

Besides antibiotin antibodies, the detection system of choice for biotin is Streptavidin or Avidin (better than antibiotin antibodies because of higher affinity).

Biotinylation of amines

Amine biotinylation was popularized with the succinimidyl esters of biotin. Uptima recommands the NHS-lc-Biotin or NHS-lc-lc-Biotin that allows generally increased detection levels.

Non sulfo agents (UP85262) may be prefered for soluble proteins if organic solvents are acceptable, because hydrolysis can be better controlled, beside cost reasons. They allow also intracellular labeling.

Sulfosuccinimidyl esters of biotin (UP54398, UP29847) are extensively used as topological probes to label proteins in the outer membrane surface (Marmorstein 1998) , or when the use of organic solvents should be avoided.

SulfoNHS-Biotins have been used for example to differentiate membranes with different polarity (Kroepf 2001, Marmorstein 1996) , and internalization of membrane proteins and cell-surface targeting of proteins (Ray 1999).

NHS-Biotin	UP39044A	100mg	SulfoNHS-Biotin	UP52117A	100mg
	UP39044B	50mg		UP52117B	50mg
A classic for biotinylation on amines Reacts with primary amines at pH7-9 Short spacer Ideal for antibody and DNA biotinylation Economic			Colorimetric quantitation of biotin Soluble directly in aqueous buffer (no need DMSO) Do not cross biological membranes / label inside cells
N-hydroxySuccinimido-Biotin MW 341.4 CAS [35013-72-0] Technical Sheet			Sulfo-Succinimido-Biotin Acid MW 443.4 CAS[119616-38-5] Technical Sheet
Applications / Literature Immunoassays Receptor studies Nucleotide labeling (Bergtstrom 1990) Ngo (1982), Gilliam (1968;1986), Wojchowski (1986), Duhamel (1985), Chichibu (1989), Nakayama (1990), Flanders (1982)			Applications / Literature Cell membrane studies Lee (1984), LaRochelle (1986), Altin(1995), Bubb (1991), LeBivic (1989), Vernachio (1989), Yeh (1996), Ingalls (1986)

NHS-Ic-Biotin	UP85262A	100mg	SulfoNHS-Ic-Biotin	UP54398A	100mg
	UP85262B	50mg		UP54398B	50mg
Extended spacer arm than NHS-Biotin for improved availability of biotin Reacts with primary amines at pH7-9 Extended spacer Ideal for antibody and DNA biotinylation Economic			The water soluble analog Soluble directly in aqueous buffer (no need DMSO) Do not cross biological membranes / label inside cells
Succinimidyl-6-(biotinamido)-hexanoate MW 455 CAS [72040-63-2] Technical Sheet Applications / Literature Solid support labeling Hydrophobic molecules labeling Receptor studies In Situ labelling Immunoassays: immunoglobulins Leary (1983), Schuberth (1996), Gretch (1987), Sorenson (1996), Foxall (1995), Hnatowich (1997), Kerstens (1995), Wheeler (1996), Green (1971), Suter (1986), Hofmann (1982), Leary (1983), Costella (1979)			Sulfo Succinimidyl-6-(biotinamido)-hexanoate MW 556.6 CAS[127062-22-0] Technical Sheet Applications / Literature Cell membrane studies In vivo targetting Suter (1986), Hnatowich (1987)

SulfoNHS-Ic-Ic-Biotin	UP29847A	50mg
	UP29847B	100mg
The longest spacer version of the NHS-x-Biotins 14 atom extended spacer! Water soluble, do not cross biological membranes
6-Biotinamidocaproylamido)caproic acid N-hydroxysuccinimide ester MW 567.7 CAS [89889-52-1] Technical Sheet Applications / Literature Special applications were NHS-lc-Biotin gives low sensitivity of detection when buried labeling sites are suspected. Almenoff (1993), hyman (1991)

NHS-imino-Biotin	UP35329A	100mg	SulfoNHS-SS-Biotin	UP53031A	100mg
	UP35329B	50mg		UP53031B	50mg
Perfect for further biotin-affinity purification (and immunoprecipitation) Lower affinity for (strept)avidins products than normal biotin Binds at alcaline pH Dissociates at pH4 N-HydroxySuccinmide immino-Biotin MW 340.4 Applications / Literature Recovery of biotinylated molecules after biotin-affinity separation from complex mixtures: plasmatic proteins bound to membrane cells, in-vivo biotinylated proteins… Suton (1984), Yamamoto (1984) , Orr (1981)			The reversible biotinylation agent Soluble directly in aqueous buffer (no need DMSO) Reacts with primary amines at pH7-9 Extented spacer and cleavable by reducing agents Do not cross biological membranes / label inside cells Sulfo-Succinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate MW 606.7 Technical Sheet Applications / Literature Conjugates for in-vivo release Recovery of non biotinylated molecules after biotin affinity purification Shimkus (1985), Duhamel (1985), Gottardi (1995), Soukup (1995), Mouton (1982), Gretch (1987), Lomont (1976) Related products see reducing agents DTT #UP28425, TCEP #UP242214

Related products			-	Fluorescent Labeling		-
MTSE-Biotin	NEW	UPR5752	10mg	FITC	UP017396	100mg
					UP01739A	1g
2-((biotinoyl)amino)ethyl MethaneThioSulfonate MW 381.52 C₁₃H₂₃N₃O₄S₃ This reagent would fit inside a cylinder about 0.6nm in diameter and 1nm in length (Akabas 1992). Half-life (pH7.0, 20°C): ca 12 min, Half-life (pH6.0, 20°C): ca 92 min, Half-life (pH7.0, 4°C): ca 116 min (Karlin 1998)				Very popular fluorescent dye Reacts with amines but also other chemical groups Purity > 80% l exc/em : 495/520 nm Fluorescein isothiocyanate MW 389.4 CAS : 3326-32-7 C₂₁H₁₁NO₅S

Buffers
Desalting products	-

Biotinylation of Thiols

Sulfhydryl biotinylation is useful for the detection or modification of SH containing sites or study SH dependant structures. It works also when SH are introduced :

into proteins (with SATA reagent UP84235)

into peptides : the introduction of Cys residue during the synthesis at the terminus of the aa chain is now a popular technology to get a site specific and oriented biotinylation

into nucleotides, that are thiol-modified for similar goals

Uptima recommands for classic biotinylation applications the maleimido-biotin (UP48198) or its more soluble maleimido-lc-Biocytin (UP99687), because of their quick, quasi stoechiometric and very specific reactivity.

Maleimido-Biotin	UP48198A	100mg	Biocytin-lc-maleimide	UP99687A	25mg
Biotinylates proteins on specific site Maleimide reacts specifically with free sulfhydryls at pH6.5-7.5 Allows more defined labeling of proteins Avoids undesired amine modification on proteins			A long spacer analog of Maleimido-Biotin Maleimide reacts specifically with free sulfhydryls at pH6.5-7.5 Biocytin binds to avidin with lower affinity than biotin does
Maleimido-biotin MW 451 Technical Sheet Applications / Literature Biotinylation of (Fab’)2 Ig fragments Biotinylation of SH of enzyme catalytic site Related Products SATA #UP84235A, Iminothiolane #UP42425A			N-Biotinyl-N-(3-MaleimidoPropionyl)-L-Lysine MW 523.6 CAS[102849-12-7] Technical Sheet Applications / Literature Protein blotting and immunoassays: detection of SH groups on dot blot in the femto range Protein immobilization Cytochemical studies Roffman (1986), Bayer (1987)