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Biotin and Streptavidin

The biotin-streptavidin complex is one of the most popular tagging systems for the conjugation of biomolecules, such as proteins, lipids and nucleic acids, as well as that of synthetic molecules, such as fluorescent labels. It has found strong success in the area of sample preparation as a core part of many purification systems and plays a critical role in many detection systems for instruments such as microscopy and flow cytometry.

 



Biotin-Streptavidin Conjugation System



Illustration of the Streptavidin-Biotin interaction. The multivalent properties of streptavidin allow it to bind up to four biotin molecules with a high degree of affinity. Biotin is typically conjugated to an enzyme, antibody or target protein.
The widespread adoption of the biotin-streptavidin conjugation system is primarily due to two factors. The first is the relatively small size of the biotin and streptavidin molecules themselves, which allows for extensive binding to biologically active macromolecules, such as antibodies, without impedance to their functions (i.e. an antibody's binding site). The second is the specificity with which biotin and streptavidin bind to each other, as well as the strength of the subsequent bond, which allows for powerful, stream-lined bioassay applications. Streptavidin tetramers have an extraordinarily high binding affinity for biotin with a dissociation constant (Kd) of approximately ~10-14 mol/L. This tight and specific binding is rapid and able to withstand extremes in pH, temperature, organic solvents, and denaturing reagents.

Advantages of the biotin-streptavidin system


A key benefit of the biotin-streptavidin system is its ability to improve detection sensitivity. This in large part is due to the tetrameric conformation of streptavidin. One streptavidin protein has the capacity to bind four biotin molecules with high affinity and selectivity. This multiplicity enables the amplification of weak signals and improves the detection sensitivity for medium- and low-abundance targets in mammalian cells or tissues with a simple workflow.

Another key benefit is the versatility of the biotin-streptavidin system. Because streptavidin can be conjugated to a variety of reporter tags, it can be readily incorporated into virtually every immunoassay. For example, enzyme conjugates of streptavidin are widley used in enzyme-linked immunosorbent assays (ELISAs), while fluorescently labeled streptavidin, such as iFluor® 488 streptavidin, are widely used in cell surface labeling, fluorescence activated cell sorting (FACS) and other fluorscence imaging applications.

Applications

 

Biotin


Biotin is a small 244 dalton hapten molecule. It's exceptionally strong binding affinity for streptavidin is commonly exploited to detect and monitor biological targets of interest. Biotin exhibits two characteristics that make it ideal for bioconjugate development. First, is biotin's relatively small size. This permits the labeling of multiple biotin tags to a single protein or antibody without significantly impeding their biological reactivity. Second, is the valeric acid side chain of the biotin molecule. This chain can be derivatized to facilitate the incorporation of various reactive groups used to chemically label biotin to proteins without altering biotin''s binding affinity for streptavidin.
AAT Bioquest offers biotinylated secondary antibodies, proteins, nucleotides and other small molecules for use in streptavidin-based amplification techniques. For assays in which a biotinylated probe is not available, we provide many biotinylation reagents and kits that enable reasearcher to chemically label proteins, nucleic acids and surface materials to make custom biotinylated reagents.

Featured Products

 

Fluorescent Biotin Derivatives


Fluorescent biotin derivatives contains both a fluorophore and a biotin moeity in the same molecule. These reagents are used for detecting and quantiting biotin-binding proteins by fluorescence. The strong quenching associated with streptavidin binding to fluorecent biotin can be used to precisely measure the concentration of streptavidin (or avidin).

 

Table 1. Available Products for Fluorescent Biotin Derivatives

Product
Ex/Em (nm)
Ext. Coeff.¹
FQY²
Unit Size
Price
Cat No.
Fluorescein biotin497/51680,0000.79005 mg$1003017
Biotin-4-fluorescein *CAS 1032732-74-3*492/51880,0000.79005 mg$1003006
Cy5 biotin conjugate650/669250,0000.275 mg$2053100
  1. Ext. Coeff. = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).

 

Biotinylated Secondary Antibodies



Immunofluorescent stain of U+03b1-tubulin in HeLa cells. U+03b1-tubulin in fixed and permeabilized HeLa cells were labeled with rabbit anti-tubulin primary antibody, followed by incubation with biotinylated goat anti-rabbit IgG (H&L) (Cat No. 16794), and then visualized with iFluor® 555-streptavidin conjugate (Cat No. 16989).
We offer biotinylated secondary antibodies to use with streptavidin-based amplification techniques. A key advantage to using a biotinylated secondary is the flexibility to use the same secondary antibody across multiple applications, by simply using a different streptavidin conjugate.

Readily available streptavidin conjugates


 

Table 2. Available Products for Biotinylated Secondary Antibodies

Product
Host/Isotype
Species Reactivity
Unit Size
Price
Cat No.
Biotinylated goat anti-mouse IgG (H+L)Goat/IgGMouse1 mg$15316729
Biotinylated goat anti-rabbit IgG (H+L)Goat/IgGRabbit1 mg$15316794

 

Biotinylated Nucleotides


Biotin-modified nucleoside triphosphate analogs, such as dUTP and dCTP, can be enzymatically incorparted into DNA or RNA fragments for use in fluorescence in situ hybridization (FISH), DNA arrays, microarrays and other hybridization techniques. Standard enzymatic non-radioactive DNA labeling reactions including 3''-end labeling, cDNA labeling, nick translation, PCR and random prime labeling. Each biotinylated nucleotide contains either an 11-, 14-, 16- or 20-atom spacer between the biotin and its attachment point on the nulceotide. This facilitates its detection and signal amplification by fluorophore and enzyme streptavidin conjugates or agarose and magnetic beads.

 

Table 3. Available Biotinylated Nucleotides

Product
Linker Length
Unit Size
Price
Cat No.
Biotin-11-dUTP11 carbons25 nmoles$10017016
Biotin-16-dUTP16 carbons25 nmoles$10017017
Biotin-20-dUTP20 carbons25 nmoles$10017018
Biotin-14-dCTP14 carbons25 nmoles$10017019

 

Biotinylated Amino Acids


Biotinylated peptides are suitable for immunodetection studies and biomedical screening assays that require immobilization onto streptavidin or avidin coated microtitre plates, beads or membranes. Biotin can be linked to the N-terminus of peptides or to the side chains of lysine (Lys), glutamic acid (Glu) or aspartic acid (Asp) during Fmoc solid phase synthesis. Incorportion of a PEG-spacer in both FMOC-Glu(biotinyl-PEG)-OH and FMOC-Asp(biotinyl-PEG)-OH reduces steric hindrance between target peptide and streptavidin, leading to better biotin binding. The hydrophilic nature of the PEG-spacer minimizes non-specific interactions and improves the solubility of the reagent and biotinylated peptide.

 

Table 4. Available Biotinylated Amino Acids

Product
Labeling Principle
Derivative
Unit Size
Price
Cat No.
FMOC-Lys(Biotin)-OH Uses FMOC-SPPS to prepare biotin-labeled peptidesLysine100 mg$1005020
FMOC-Glu(biotinyl-PEG)-OH Uses FMOC-SPPS to prepare biotin-labeled peptidesGlutamic acid100 mg$2055026
FMOC-Glu(biotinyl-PEG)-OH Uses FMOC-SPPS to prepare biotin-labeled peptidesGlutamic acid1 g$9985027
FMOC-Asp(biotinyl-PEG)-OH Uses FMOC-SPPS to prepare biotin-labeled peptidesAspartic acid100 mg$2055023
FMOC-Asp(biotinyl-PEG)-OH Uses FMOC-SPPS to prepare biotin-labeled peptidesAspartic acid1 g$9985024

 

Reactive-Biotin Derivatives


Reactive biotin derivatives contain reactive moieties, such as succinimidyl esters or maleimides, that facilitate the labeling of biotin onto proteins, antibodies and other macromolecules. When selecting a reactive biotin derivative, consider the following:
  • Funtional group targeting - specific reactive moieties target and bind to their respective functional groups for targeted or non-selective biotinylation of proteins and other macromolecules (e.g. succinimidyl esters target primary amines and maleimides target thiol or sulfhydryl groups).
  • Solubility - this influences biotinylation of the target protein or macromolecule.
  • Spacer Arm Length (PEG) - enhance detection senstivity of the target protein and reduces steric hindrance.

ReadiView™ Biotinylation Reagents



ReadiView™ biotin succinimidyl ester (Cat No. 3059) conjugated with specially designed color tag (CT) for easy determination of biotinylation.
Determining a protein's degree of biotinylation is quite challenging because the intrinsic absorption of biotin is difficult to discern from proteins and nucliec acids. To facilitate this determination, we offer a series of chromophoric reactive boitin derivatives, ReadiView™ biotin.

These novel biotinylation reagents contain a specially designed 'Color Tag (CT)' optimally positioned between two spacer arms. These spacer arms reduce steric hindrance and improve water solubility, while the CT tag makes the degree of biotinylation readily quantifiable by calculating the absorption ratio of 280 nm/385 nm. The CT tag does not affect biotins complexation with streptavidin and has minimal quenching affect on fluorescent streptavidin conjugates. ReadiView™ biotin is availble in amine-reactive, thiol-reactive and click-chemistry formats.

 

Table 5. Available Reactive Biotinylation Reagents

Product
Chemical Reactivity
Reactive Moiety
Unit Size
Cat No.
Biotin, succinimidyl ester *CAS 35013-72-0*AmineSuccinimidyl Ester100 mg3002
Biotin-X, succinimidyl ester *CAS 72040-63-2*AmineSuccinimidyl Ester25 mg3010
Biotin PEG2 succinimidyl esterAmineSuccinimidyl Ester25 mg3016
Biotin PEG4 succinimidyl esterAmineSuccinimidyl Ester25 mg3022
Biotin C2 maleimideThiolMaleimide25 mg3005
Biotin PEG2 maleimide *CAS 305372-39-8*ThiolMaleimide5 mg3015
Biotin PEG2 amine *CAS 138529-46-1*Aldehyde, Carboxylic Acid, KetoneAmine5 mg3014
Biotin PEG3 amineAldehyde, Carboxylic Acid, KetoneAmine5 mg3024
Biotin cadaverineAldehyde, Carboxylic Acid, KetoneCadaverine5 mg3004
Biotin ethylenediamine *CAS 216299-38-6*Aldehyde, Carboxylic Acid, KetoneEthylenediamine 5 mg3003

Table 6. Available ReadiView™ Biotin Products

Product
Chemical Reactivity
Spacer
Unit Size
Price
Cat No.
ReadiView™ biotin succinimidyl esterAminesPegylated5 mg$1003059
ReadiView™ biotin maleimideThiol or sulfhydryl groupsPegylated5 mg$2053058
ReadiView™ biotin hydrazideAldehyde, Carboxylic acid, KetonePegylated5 mg$2053055
ReadiView™ biotin amineCarbonylPegylated5 mg$2053053
ReadiView™ biotin acidAminePegylated5 mg$1003050

 

Enzyme-Labeled Streptavidin Conjugates



IHC detection of EpCAM in FFPE lung adenocarcinoma tissue. Human adenocarcinoma tissue sections were incubated with rabbit anti-EpCAM primary antibody followed by biotyinylated goat-anti rabbit IgG (H&L). The samples were then incubated with HRP-streptavidin, and the signal was developed using ReadiUse™ Stayright™ Purple.
Enzyme-labeled streptavidin conjugates are commonly used in enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunoblotting, and in situ hybridization techniques. Enzymes horseradish peroxidase (HRP) and alkaline phosphatase (ALP) are extensively used as reporter tags because of their high turnover rate, stability, ease of conjugation and low cost.

To maximize retention of both enzyme and protein activity, our HRP-streptavidin and AP-streptavidin conjugates are prepared at a 1:1 (enzyme to streptavidin) labeling ratio. Chromogenic, fluorogenic and chemiluminescent stubstrates are available for these enzyme conjugates.

Learn More

 

Table 7. Available Enzyme-labeled Streptavidin Conjugates

Product
Enzyme
Unit Size
Cat No.
HRP-streptavidin conjugateHorseradish peroxidase (HRP)1 mg16920
AP-streptavidin conjugateAlkaline phosphatase (AP)1 mg16921

Table 8. Available HRP Substrates

Product
Detection
Abs/Ex (nm)
Ex (nm)
Unit Size
Cat No.
ReadiUse™ ABTSChromogenic420 1 L11001
ReadiUse™ TMBChromogenic650 100 mL11012
ReadiUse™ TMBChromogenic650 1 L11003
Amplite® ADHPFluorogenic57158425 mg11000
Amplite® BlueFluorogenic32440925 mg11005
Amplite® RedFluorogenic5715841000 Assays11011
Amplite® IRFluorogenic6486681 mg11009
LuminolChemiluminescent3554121 g11050

Table 9. AP-Streptavidin Conjugate & Substrates

Product
Detection
Abs/Ex (nm)
Ex (nm)
Unit Size
Cat No.
pNPP *CAS 4264-83-9*Chromogenic405 25 mg11619
FDP *CAS 217305-49-2*Fluorogenic4985175 mg11600
MUP, disodium salt *CAS 22919-26-2*Fluorogenic36044825 mg11610
MUP, disodium salt *CAS 22919-26-2*Fluorogenic36044810 g11612
DiFMUPFluorogenic3584505 mg11627
SunRed™ PhosphateFluorogenic6536615 mg11629
D-Luciferin phosphate *CAS 145643-12-3Chemiluminescent  5 mg12512

 

Fluorescent Streptavidin Conjugates



Immunofluorescent stain of &alpha-tubulin in HeLa cells. &alpha-tubulin in fixed and permeabilized HeLa cells were incubated with rabbit anti-tubulin primary antibody, followed by incubation with biotinylated goat anti-rabbit IgG (H&L) (Cat No. 16794), and then visualized with iFluor® 647-streptavidin conjugate (Cat No. 16966. Nuclei were counterstained using DAPI (Cat No. 17507.)
Fluorescent streptavidin conjugates are widely used in fluorescence imaging to detect biotinylated antibodies, ligands and DNA probes for in situ hybridization techniques, immunohistochemistry and multicolor flow cytometry. We offer a comprehensive portfolio of fluorescent streptavidin labeled with iFluor® dyes, mFluor™ dyes, phycobiliproteins and tandem dyes, and more. Benefits of our iFluor® streptavidin conjugates include exceptionally bright fluorescence, excellent photostability and pH insensitive fluroescence over a wide molar range.

iFluor® Streptavidin Conjugates


Streptavidin conjugates labeled with iFluor® dyes exhibit exceptionally bright and photostable fluorescence, outperforming Alexa Fluor® and other spectrally similar conjugtes. iFluor® streptavidin conjugates are designed for use in a variety of cell analysis and protein analysis applications including imaging, flow cytometry and fluorescent western blotting. As a precaution, avoid using blue fluorescent streptavidin conjugates when detecting low abundance targets. Blue fluorescent dyes have lower fluorescence and can experience higher non-specific background than other dyes.

Learn More


 

Table 10. iFluor-Streptavidin Conjugates For Imaging and Flow Cytometry

Product
Ex/Em (nm)
Filter Set
Ext. Coeff.¹
FQY²
Unit Size
Price
Cat No.
iFluor® 350 Streptavidin 344/448DAPI20,0000.951 mg$15316980
iFluor® 405 Streptavidin402/425DAPI37,0000.911 mg$15316982
iFluor® 488 Streptavidin491/516FITC75,0000.91 mg$15316985
iFluor® 514 Streptavidin527/554TRITC80,0000.831 mg$15316986
iFluor® 532 Streptavidin543/563TRITC90,0000.681 mg$15316987
iFluor® 546 Streptavidin541/557TRITC100,0000.67200 µg$7916958
iFluor® 555 Streptavidin556/569TRITC100,0000.641 mg$15316989
iFluor® 568 Streptavidin568/587TRITC100,0000.57200 µg$7916960
iFluor® 594 Streptavidin587/603Texas Red180,0000.531 mg$15316992
iFluor® 633 Streptavidin638/652Texas Red250,0000.291 mg$15316995
  1. Ext. Coeff. = Extinction coefficient at their maximum absorption wavelength. The units of extinction coefficient are cm-1M-1.
  2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).
  3. N/D = Not determined.

 

mFluor™ Streptavidin Conjugates for Flow Cytometry



Flow cytometry analysis of HL-60 cells stained with (Red) or without (Green) 1ug/ml Anti-Human HLA-ABC-Biotin and then followed by mFluor™ Blue 570-streptavidin conjugate (Cat No.16935).
Streptavidin conjugates labeled with mFluor™ dyes exhibit minimal self-quenching, producing intensely fluorescent and photostable conjugates for flow cytometery. mFluor™ streptavidin conjugates are designed for optimal excitation by one of the major laser lines commonly equipped in flow cytometers, such as the 405 nm, 488 nm, 532 nm, 561 nm or 633 nm laser lines.

Learn More

 

Table 11. mFluor™ Streptavidin Conjugates for Flow Cytometry

 

Streptavidin APC, PE, PerCP and Tandem Dye Conjugates for Flow Cytometry


Streptavidin PE, APC and PerCP conjugates are commonly used in applications that require high sensitivity, but not phostostability, primarily flow cytometry, FACS, immunophenotyping, and microarrays. The unusually bright fluorescence of phycobiliproteins is due to their unusually high extinction coefficients and quantum yields.

Tandem dye conjugates compromise of a donor phycobiliprotein (e.g. APC or PE) labeled to acceptor iFluor® dyes that emit fluorescence at a longer-wavelength. Using fluorescence resonance energy transer (FRET), light absorbed by the the donor phycobiliprotein results in fluorescence emission of the acceptor. In flow cytometry, tandem dyes are ideally suited for multicolor parametric analysis of cells due to their exploitation of a single excitation source and their significantly large Stokes shifts.

 

Table 12. Streptavidin APC, PE, PerCP and Tandem Dye Conjugates for Flow Cytometry

Streptavidin Conjugates
Ex (nm)
Em (nm)
Filter Set
Cat No.
RPE Streptavidin564574575/2616900
APC Streptavidin651660660/2016902
PerCP Streptavidin477678695/4016905
RPE-iFluor® 647 Streptavidin568666670/1416906
RPE-iFluor® 750 Streptavidin566778780/6016907
APC-iFluor® 750 Streptavidin651791780/6016908
RPE-Cy7 Streptavidin566778780/6016916

 

Streptavidin-Xtra™ Conjugates



Comparison of HeLa cells stained with Streptavidin-Xtra™ IF488 and Streptavidin-Alexa Fluor® 488. HeLa cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.02% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 1 µg/mL U+03b1-tubulin Mouse mAb for 1 hour at room temperature. Biotinylated goat anti-mouse IgG (H+L) (Cat No. 16729) was used for detection U+03b1-tubulin and visualized with Streptavidin-Xtra™ iFluor 488 (Cat No. 46001) and Streptavidin-Alexa Fluor® 488. Cells were counterstained with Hoechst 33342 (Cat No. 17535).
The optimal degree of labeling of Streptavidin-Xtra™ iFluor® conjugates produce the brightest fluorescent streptavidin conjugates for the detection of biotinylated antibodies in cell imaging, flow cytometry and western blot applications. Streptavidin-Xtra™ iFluor® conjugates image low-abundance targets with greater sensitivity and detail compared to traditional streptavidin Alexa Fluor® conjugates.

Advantages

  • Bright and photostable fluorescence without significant self-quenching
  • High siganl-to-noise ratios: 3~5 fold signal improvement versus Alexa Fluor®
  • pH insensitive fluroescence over a wide molar range

 

Table 13. Streptavidin-Xtra™ Conjugates

Streptavidin Conjugates
Ex (nm)
Em (nm)
Filter Set
Cat No.
Streptavidin-Xtra™ iFluor® 488491516FITC46001
Streptavidin-Xtra™ iFluor® 555556569TRITC46003
Streptavidin-Xtra™ iFluor® 594587603Texas Red46005
Streptavidin-Xtra™ iFluor® 647654669Cy546007

 

Product Ordering Information



 

Table 14. Ordering Info for Biotin Products

Cat#
Product Name
Unit Size
Price
Ex
Em
3001Biotin *CAS 58-85-5*1 g79
3002Biotin, succinimidyl ester *CAS 35013-72-0*100 mg205
3003Biotin ethylenediamine *CAS 216299-38-6*10 mg100
3004Biotin cadaverine25 mg100
3005Biotin C2 maleimide25 mg310
3006Biotin-4-fluorescein *CAS 1032732-74-3*5 mg100492518
3007Biotin hydrazide *CAS 66640-86-6*25 mg79
3009Biotin-X NTA [Biotin-X nitrilotriacetic acid, potassium salt] *CAS 856661-92-2*1 mg100
3010Biotin-X, succinimidyl ester *CAS 72040-63-2*25 mg79
3014Biotin PEG2 amine *CAS 138529-46-1*5 mg100

Table 15. Ordering Info for Streptavidin Products

Cat#
Product Name
Unit Size
Price
16900RPE-streptavidin conjugate100 ug100
16901RPE-streptavidin conjugate1 mg310
16902APC-streptavidin conjugate100 ug153
16905PerCP-streptavidin conjugate100 ug205
16906RPE-iFluor® 647-streptavidin conjugate100 ug205
16907RPE-iFluor® 750-streptavidin conjugate100 ug205
16908APC-iFluor® 750-streptavidin conjugate100 ug205
16910FITC-streptavidin conjugate1 mg153
16911Texas Red®-streptavidin conjugate1 mg153
16912Cy3®-streptavidin conjugate1 mg153