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SKU:SA1072
Clonality:Polyclonal
Application:IHC, ICC
Price: $300.00
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Product Overview

Product Name Cy3 Conjugated anti-Mouse IgG SABC Kit
Description Cy3 Conjugated Anti-Mouse IgG SABC Kit is a four-component system for indirect sensitive immunofluorescent detection of proteins on tissue sections via ICC, IHC-P or IHC-F probed with mouse primary antibody by utilizing a biotinylated secondary antibody that links target-bound primary antibodies to streptavidin-Cy3 conjugate forming a stable StreptAvidin-Biotin Complex (SABC) .
Synonyms Cy3 Conjugated Anti-Mouse IgG StreptAvidin-Biotin Complex Kit; Cy3 Conjugated SABC Kit; Goat Anti-Mouse IgG SABC Kit, Cyanine 3 Conjugated; Cy3-conjugated streptavidin, Biotin-conjugated goat anti-mouse IgG Protein staining kit
Reagent Type Protein assay kit / Secondary detection reagent kit
Immunoreagent Secondary antibody
Reporter Cy3 (Cyanine 3) fluorescent dye
Reporter-carrying Molecule Streptavidin
Antibody Conjugate “Long-arm” biotin: Biotinamidohexanoic acid N-hydroxysuccinimide ester, CAS# 72040-63-2
Antibody Class IgG
Host Goat
Target Species Mouse
Immunogen Whole molecule mouse IgG
Specificity Mouse IgG specific

*Reactive against the heavy chains of mouse IgG and the light chains common to most mouse immunoglobulins, but not against non-immunoglobulin serum proteins

Cross-reactivity No cross-reactivity with human/bovine/rabbit IgG; may cross-react with immunoglobulins from other species
Usage Indirect immunofluorescent staining assays
Validated Applications Immunohistochemistry (paraffin-embedded (IHC-P) and frozen(IHC-F) sections), Immunocytochemistry (ICC)

*Our Boster Guarantee covers the use of this product in the above tested applications.

Storage At 4•C for one year; avoid freezing
Precautions FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC AND CLINICAL USE

Kit Components

Kit Component Component Description Concentration Size
Normal goat serum blocking reagent For the block of tissue sections 10x 5 ml
Biotinylated Secondary Antibody Goat Anti-mouse IgG
   •   Polyclonal
   •   Affinity purified
   •   Solid phase adsorbed against human serum proteins
   •   Labeled with “long-arm” biotin (Biotinamidohexanoic acid N-
        hydroxysuccinimide ester, CAS# 72040-63-2)
2 mg/ml, 100x 0.5 ml
Cy3 conjugated streptavidin Manufactured by Boster’s proprietary method, the complex is very stable and offers superior amplification of the antigen signals 1 mg/ml, 100x 0.5 ml
Three drop bottles For dilution use - -

*Additional components can be purchased. If you need extra of the above components please order them together to avoid additional shipping charges.

Assay Information

Assay Type Immunoanalytical
Detection Method Fluorescent
Amax / Emax 554 nm / 568-574 nm
Assay Purpose Protein detection and/or quantification
Technique StreptAvidin-Biotin complex (SABC)
Sample Type Mouse primary-antibody-probed:
   •  Formalin-fixed paraffin-embedded (FFPE) tissue sections on slides (IHC-P)
   •  Thawed frozen samples (IHC-F)
   •  Cultured cells
   •  Freshly prepared blood smears
Recommended Working Concentration See protocol in Datasheet for each application
Equipment Needed Excitation light source;
Filter set and detector: fluorescence microscope (can be combined with confocal microscope), fluorescence plate-reader;
Additional Materials Required Primary antibody against target antigen raised in mouse;
Diluent Buffer (0.01M PBS);
Antigen Retrieval Buffer (0.01 M Citrate Buffer);
0.1% trypsinase or compound digest solution (Catalog number: AR0022);
Other application-specific reagents and appliances;

Main Advantages

High Specificity High signal-to-noise ratio ensured by the specificity of recognition of:
   •  Antigen - primary antibody
   •  primary-secondary antibody
   •  biotin-streptavidin
Outstanding Sensitivity Extremely high extinction coefficient and good quantum yield of Cy3 result in brighter fluorescence and less background compared to conventional dyes with similar spectral properties (TRITC); easily detected by naked eye on gels and in solution; especially suitable for very sensitive localization assays
High Signal Amplification Multiple Cy3 molecules localize at the antigenic site via:
   •  Multiple secondary antibodies binding to a single primary antibody
   •  Multiple biotin molecules conjugated to a single secondary antibody
   •  Multiple Cy3 molecules conjugated to a single streptavidin molecule
Robust High specificity at high dilutions
Fast    •  Fewer number of processing steps - no need for adding a substrate
   •  Less optimization required compared to enzymatic detection
   •  Generates strong signals in a relatively short time span
   •  Signal can be observed directly
Biochemical Stability    •  Exceptional stability of biotin-streptavidin binding: streptavidin-biotin complex is highly resistant to organic solvents, denaturants (e.g. guanidinium chloride), detergents (e.g. SDS, Triton), proteolytic enzymes, and extremes of temperature and pH; additional chemical modifications (e.g. reporter and/or antibody conjugation) do not affect biotin-streptavidin binding affinity;
   •  Cyanine dyes are better able to withstand the harsh dehydration and embedding conditions required for mounting sections
Excellent Photostability    •  Reduced fluorescence quenching - sulfonated cyanines are less prone to aggregation in water due to reduced dye-dye interactions;
   •  Cy3 conjugates are more photostable in the aqueous environment, as well as in non-polar media compared to other orange-red fluorescing dye conjugates
Absolute Quantification Precise quantitative analysis of fluorescence microscopy images can provide absolute protein amount and information regarding stoichiometry of protein complexes
Superb Signal Detection Sharp signal development
Precise signal detection
Explicit target localization
Flexible No need to label each antibody against each target protein with a fluorescent dye - use fluorescently labeled 2ndary antibodies against target species
Sterically Optimal    •  Small size of biotin does not interfere with antibody binding;
   •  Streptavidin-biotin complex has optimal size allowing for cell and tissue penetration
Instrument and Protocol Compatibility Excitation and emission spectra correspond with filter sets and laser settings of all popular fluorescence instrumentation allowing for easy adjustment of an existing protocol
Multiplex Multiplex Compatibility Compatible with colocalization studies (multiple antigens concurrent detection) even in close proximity using primary antibodies from different host species for simultaneous detection by fluorophore-conjugated secondary antibodies, or using multiple differently colored fluorophores (e.g. Cy3 and FITC and TRITC) in the same experiment for target differentiation.

Conjugates Information

Cy3

Conjugate Name Cy3 (Cyanine 3)
Molecular Weight ~730 g/mol
Fluorescence spectrum*
Excitation Max 554 nm
Emission Max 568-574 nm
Color Bright orange-red
Excitation Laser Line 488 nm; 514 nm ; 532 nm; 543 nm
Argon laser: 514 nm/ 528 nm lines - 50% excitation ;
Helium/neon laser : 543 nm line - 75% excitation;
Mercury lamp: 546 nm line - 75% excitation
Filter Set 530/30
Fluorescence Channel FL2,FL4,PM1
Stokes shift 20 nm
Molar Extinction Coefficient 150 000 / 136 000 M-1cm-1
Quantum Yield 0.04, / 0.15
Solubility Good water solubility due to sulfonation
Fluorescence Lifetime < 0.3 ns
pH Sensitivity No effects on the spectral characteristics have been observed under a range of pH conditions
Chemical Reactivity N-hydroxysuccinimide (NHS)-activated form: reactive towards exposed N-terminal α-amino groups and the ε-amino groups of lysine residues to form stable amide bonds

Biotin

Names Trivial Names Long Arm Biotin
Synonyms Succinimidyl-6-(biotinamido)hexanoate; NHS-LC-Biotin;
NHS-X-Biotin; Biotin-LC-NHS; LC NHS-(+)-Biotin; BIOTIN-X, SUCCINIMIDYL ESTER;
Biotinamidohexanoic acid, N-hydroxysuccinimide ester
IUPAC Name 6-[[5-[(3aS,4S,6aR)-hexhydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]-1-oxopentyl]amino]-2,5-dioxo-1-pyrrolidinyl ester-hexanoic acid
Molecular Formula Structural Formula
Chemical Formula C20H30N4O6S
Identifiers CAS Number 72040-63-2
Properties Molecular Weight 454.55
Spacer length 22.4 Å
Appearance A crystalline white solid
Melting point 160-162 ˚C
Solubility in water ≤2 mg/mL with sonication
Solubility in organic solvents Soluble in DMF, DMSO and Methanol
Description & Usage NHS-LC-Biotin (NHS-X-Biotin) is a long chain amine reactive biotinylation reagent. It is used to attach biotin to primary amines under alkaline conditions (pH~8-9). NHS-LC-Biotin contains a biotin moiety, an elongated alkyl chain spacer arm (~22 Å) and an amine-reactive active NHS ester. NHS-LC-Biotin reacts efficiently and irreversibly with a wide range of compounds containing primary amine (-NH2) residues, including proteins, antibodies and other biomolecules (e.g. lysines on the surface of proteins). The spacer arm reduces steric hindrance when binding several biotinylated molecules to one avidin complex. This biotinylation compound is membrane permeable (contains no charged groups), making it well suited for intracellular protein labeling.
Protein Biotinylation Mechanism

Streptavidin

CAS Number 9013-20-1
Molecular Weight (kDa) 53
Biotin-binding Sites 4
Isoelectric Point (pI) 6.8 - 7.5
Specificity of Biotin binding High
Affinity for Biotin (Kd) ~10-14 to 10-15M
Nonspecific Binding Low
Description A tetrameric protein used to visualize biotin conjugated molecules in immunoassays

Background

SABC (StreptAvidin-Biotin Complex) kit is an immunodetection system that utilizes a reporter-labeled streptavidin and a biotinylated-secondary antibody to localize a target protein at tissue section, blotting membrane or ELISA plate previously recognized by a primary antibody. It is specially designed for displaying the localization and distribution of antigens in tissues and cells in immunochemistry and other immunodetection assays. In this technique streptavidin-biotin binding is used as a bridge between antibodies and reporters like enzymes (HRP, AP), fluorophores, chromophores, etc (Labeled Streptavidin Biotin (LSAB) method).
Streptavidin is a 47,000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin has extraordinarily strong affinity to biotin molecules. The dissociation constant (Kd) of the biotin-streptavidin complex is on the order of ≈10−15 mol/L, a million times higher than the typical affinity between antigens and their antibodies. In this method multiple biotin molecules are conjugated to a single secondary antibody and multiple reporters are conjugated to a single streptavidin, hence a single primary antibody is ultimately associated with multiple reporter molecules . Thus, by increasing the concentration of reporters at the antigenic site (increased reporter-to-antibody ratio) signal intensity and detection sensitivity are considerably amplified as compared to the detection via directly reporter-conjugated secondary antibodies. This method has also the advantage of good tissue penetration due to its optimal size compared to the ABC method where free streptavidin molecule is used as a bridge between a biotinylated antibody and a biotinylated reporter which results in an extended polymeric structure usually unable to penetrate tissue. Furthermore, streptavidin has very low non-specific electrostatic binding to tissues and cells, due to its nearly neutral isoelectric point (IP=6.0~6.5). In addition, streptavidin does not contain carbohydrate groups (opposed to avidin) which prevents non-specific binding to tissue lectins or lectin-like molecules found naturally in the kidney, brain and liver. Therefore, immunohistochemical analyses based on streptavidin-biotin complex (SABC) have extremely low background. In brief, SABC offers high specificity, high sensitivity, low background and ease-of-use.
Cyanine dyes or simply Cyanines(Cy) used as reporters here, are fluorescent molecules containing two aromatic N-heterocyclic units (indole, benzoxazol, benzothiazol), connected with a polymethine bridge with a varying number of methine units. Depending on the structure (methine units number variation, benzo-fusion, etc.), they cover the spectrum from IR to UV. . Modifications at the heterocyclic unit such as methyl, ethyl or butyl substituent, carboxyl, acetylmethoxy, and sulfo groups are used to confer hydrophilicity. For the purpose of antibody labeling Cyanines are made chemically reactive towards proteins by esterification at the N atom-side chain: reactivity towards primary amine residues by N-hydroxysuccinimide (NHS)-esterification, or towards sulfhydryl (-SH) groups of cysteine residues by maleimide esterification. Cyanines replace advantageously conventional dyes such as Fluorescein(FITC) and rhodamines (TRITC, RRX), yielding brighter and more stable fluorescence.
Cy3 is a cyanine dye with 3 carbon atoms/methine groups between two indolenine groups, NHS-ester (reactive toward primary amines), sulfonated at both indoles (hydrophilic). It fluoresces greenish yellow with excitation max at ~550 nm and emission at ~570 nm. Cy3 can be detected by various fluorometers, imagers, and microscopes with standard filter sets for Tetramethylrhodamine (TRITC) due to their nearly identical excitation and emission spectra. Due to inherently high extinction coefficient, this dye is also easily detected by naked eye on gels, and in solution. Cy3 can be excited to about 50% of maximum with an argon laser (514 nm or 528 nm lines), or to about 75% of maximum with a helium/neon laser (543 nm line) or mercury lamp (546 nm line). For double labeling Cy3 has been typically used with Cy5, with fluorescein (using narrow band-pass emission filter to minimize Cy3 fluorescence in the FITC filter set), and also with Alexa Fluor 647 for multiple labeling when using a confocal microscope. Analog of Cy3 with greater photostability and higher fluorescence intensity suited for various biological applications such as imaging and flow cytometry, is Alexa Fluor 555 dye.

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