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Secondary Antibodies


 

Secondary Antibody Overview


Secondary antibodies bind to the primary antibody to assist in detection, sorting and purification of target antigens. To enable detection, the secondary antibody must have specificity for the antibody species and isotype of the primary antibody being used and generally is conjugated.

Secondary antibodies are used throughout various types of assays, including ELISA or Western Blot, Immunohistochemistry, Flow Cytometry.  The secondary antibody type is selected according to the class of the primary antibody (e.g., IgG or IgM), the source host, and the kind of label which is preferred.   Most primary antibodies are of the IgG class and are produced in a common set of host species that includes rabbit, mouse, goat or chicken.  Therefore, anti-mouse IgG, anti-rabbit IgG, anti-goat IgG or anti-chicken polyclonal antibodies are often used. 

Choosing a secondary antibody is straightforward:  select a secondary antibody that recognizes the host species used to produce the primary antibody of interest, i.e. choose an anti-mouse secondary antibody to detect a mouse monoclonal primary antibody.  However, identifying the optimal secondary antibody requires knowledge of the detection assay.   For example:  Western blot and ELISA can be performed using colorimetric, chemiluminescent, and fluorescence reporter systems, while immunofluorescence and flow cytometry are generally limited to fluorescent reporter labels. In all of these instances, a conjugated secondary antibody is required.

 
 
view-secondary   
 

 

How to Select the Optimal  Secondary Antibody


The following variables should be considered in the approximate order of importance:

 

Secondary Antibody Selection

Criteria   Note
Host   Secondary antibody should specifically detect the species that produced the primary antibody
Polyclonal or Monoclonal   

Polyclonal antibodies are sufficient for most needs. 

 

Monoclonal antibodies are more difficult to produce but are highly specific and yield more consistent results over time. 

Detection Systems and Protocols    Does detection system use color (i.e. by eye), light (i.e. ELISA) or fluorescence (i.e. immunofluorescence)?  
Other Specificities   Subclass, Fab or F(ab’)2 fragments, reduced cross reaction to other species
 

 

2015 Secondary Side Panel

 

 

 

 

Phospho Related Antibodies

 

 

 

TrueBlot IP/Western Blot Mouse Rabbit Goat

 

 

 

The Rockland Advantage Badge

 


 

Featured Antibodies

Secondary Antibody 

Unconjugated

Fluorescein

Biotin

Alkaline
Phosphatase

 

Peroxidase

Anti-HUMAN IgG (H&L) (GOAT) Antibody
(Min X Bv Ch Gt GP Ham Hs Ms Rb Rt &
Sh Serum Proteins)
 

 

609-101-123   609-102-123   609-106-123   609-105-123   609-103-123

Anti-MOUSE IgG (H&L) (GOAT) Antibody  

 

610-1102   610-1202   610-1602   610-1502   610-1302  

Anti-MOUSE IgG (H&L) (GOAT) Antibody
(Min X Bv Ch Gt GP Ham Hs Hu Rb Rt &
Sh Serum Proteins)
  

 

610-101-121   610-102-121 610-106-121 610-105-121 610-103-121

Anti-MONKEY IgG (gamma chain) (GOAT) Antibody

 

617-101-012 617-102-012 617-106-012 617-105-012 617-103-012

Anti-RABBIT IgG (H&L) (GOAT) Antibody  

 

611-1102   611-1202   611-1602   611-1502   611-1302  

Anti-RABBIT IgG (H&L) (GOAT) Antibody
(Min X Bv Ch Gt GP Ham Hs Hu Ms Rt &
Sh Serum Proteins)
 

 

611-101-122 611-102-122 611-106-122 611-105-122   611-103-122  

Anti-SHEEP IgG (H&L) (RABBIT) Antibody

 

613-4102   613-4202   613-4602 613-4502   613-4302

F(ab')2 Anti-HUMAN IgM Fc5µ (GOAT) Antibody

 

 

709-1131   709-1231 709-1631 709-1531   709-1331  

  

 


 

Host


Attention should be paid to the host species that was used to generate the primary antibody.    Researchers should select a secondary antibody specific for detection of the primary antibody species.   For example, when using a polyclonal antibody produced by rabbit you will select an anti-rabbit secondary antibody that was raised in an alternate host species such as goat or donkey.  Rockland offers a wide range of secondary options for almost every possible experimental protocol.

 
 

 

 


 

Detection Systems and Protocols


For commonly used techniques such as Western blot and ELISA, an enzyme conjugated secondary is most likely the best choice.   Good examples are Peroxidase or Alkaline phosphatase.   (*Enzymes require a substrate for generation of signal output).  In the case of immunoassays such as immunofluorescence microscopy or flow cytometry (also called FACS) it is more typical to use a secondary antibody conjugated to a fluorochrome (i.e. FITC, DyLight™ or Cy™ dye). 

For immunoprecipitation experiments a special product that does not detect the precipitating antibody is essential for best results.  Rockland’s TrueBlot® products are useful for the accurate detection of secondary antibodies used for immunoprecipitation followed by western blot.  TrueBlot® products help eliminate heavy/light chain blotting and contamination from immunoprecipitation that can obscure target detection.  TrueBlot® products offer increased sensitivity, less background noise, and enhanced accuracy for numerous applications. TrueBlot® reagents are available in several options, including individual IP Beads and complete IP/Western Blot kits from goat, mouse, rabbit or sheep.

Enzymatic Secondary Antibody Conjugates

Reporter enzymes are used extensively in molecular biology, allowing visualization or detection of immune complexes. Horseradish Peroxidase (HRP) is a widely used reporter enzyme, and depending on the substrate it can yield a chromogenic, or luminescent product. Alkaline phosphatase is also used, most typically as the reporter in chromogenic western blot assay format.

Fluorescent Secondary Antibody Conjugates 

Rockland conjugates a broad group of secondary antibodies to many of the classic and next generation of fluorescent markers including fluorescein, Texas Red, and Phycoerythrin. All of the conjugates are ideal for various immunofluorescence based assays including fluorescent western blotting, immunofluorescence microscopy, FLISA, and more. Rockland also produces many next generation fluorochrome dyes designed for detection of primary antibodies in multiplex, multi-color analysis. Next generation fluorochrome conjugates (Atto-tec dyes, DyLight™ dyes) offer superior absorption (high extinction coefficient), high fluorescence quantum yield, and superior high photostability. 

Secondary Antibody by Applications

Secondary antibodies ELISA
Secondary antibodies Western Blot analysis
Secondary antibodies Immunostaining
Secondary antibodies Immunohistochemistry
Secondary antibodies Immunocytochemistry
Secondary antibodies Flow Cytometry

 


  

Other Specificities: Clonality, Class, Subclass, Antibody Fragments and
Pre-absorbtion


Polyclonal antibodies generated in rabbit, goat, donkey, or chicken and are usually IgG isotype and thus the secondary antibody should then be an anti-IgG H&L (i.e. both heavy and light chain specific) antibody.   IgM is also used, but at less frequency.  In certain experiments a pre adsorbed secondary antibody is needed (See Pre-Absorbed Antibodies below).  

Monoclonal primary antibodies are commonly raised in mouse, rat and Armenian hamster, although rabbit and human derived monoclonal antibodies are also popular.   The main antibody classes are designated IgA (α), IgD (δ), IgE (ε), IgG (γ) and IgM (μ).

Mouse immunoglobulin classes and subclasses:
- Classes:  IgG, IgM
- Subclasses:IgG1, IgG2a, IgG2b, IgG3; Types: κ light chain, λ light chain

Rat immunoglobulin classes and subclasses:
- Classes: IgG, IgM
- Subclasses IgG1, IgG2a, IgG2b , IgG2c;  Types: κ light chain, λ light chain

Human immunoglobulin classes and subclasses
- Classes: IgG, IgM, IgA, IgD
- Subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2; Types κ light chain, λ light chain


 

F(ab’)2 Fragment Secondary Antibodies

 

F(ab')2 fragment secondary antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact the hinge region. F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa.   Rockland offers fragmentation as a custom service.

 

 F(ab')2 fragment antibodies eliminate non-specific binding between the Fc portions of antibodies and the Fc receptors on cells.   When working with tissues or cells that have Fc receptors (spleen, blood, hematopoietic cells, leukocytes, etc.), choose an F(ab')2 fragment to eliminate non-specific binding to Fc receptors present on many cells.  F(ab')2 fragment conjugated secondary antibodies are ideal for Flow Cytometry, Immunohistochemistry and Immunofluorescence.

 

 

Fab 2

 

 

Schematic of F(ab’)2 antibody fragment.   Heavy and light chain are indicated by the H and L, disulfide bonds are yellow

 

 

 F(ab')2 Anti-RABBIT IgG [H&L] (GOAT) Antibody    711-101-002
 F(ab')2 Anti-RABBIT IgG [H&L] (GOAT) Antibody Peroxidase Conjugated   711-103-002
 F(ab')2 Anti-MOUSE IgG (H&L) (GOAT) Antibody   710-1102
 F(ab')2 Anti-MOUSE IgG [H&L] (GOAT) Antibody Biotin Conjugated Min X HUMAN Serum Proteins   710-106-019
 F(ab')2 Anti-MOUSE IgG (H&L) (RABBIT) Antibody Min X Human Serum Proteins   710-4120
 F(ab')2 Anti-MOUSE IgG (H&L) (RABBIT) Antibody Biotin Conjugated Min X Human Serum Proteins   710-4620

 F(ab')2 Anti-GOAT IgG [H&L] (RABBIT) Antibody Alkaline Phosphatase Conjugated

  705-405-002
 F(ab')2 Anti-RAT IgG [H&L] (RABBIT) Antibody Biotin Conjugated   712-406-002

 


 

Fab Fragment Secondary Antibodies


Fab fragment secondary antibodies are generated by papain digestion of whole IgG antibodies to remove the Fc region entirely generating a monovalent antibody.  Fab fragments have one antigen-binding F(ab) domain and therefore are monovalent with a molecular weight of about 50 kDa. 


 

Affinity Purified and Pre-Adsorbed Antibodies


Secondary antibodies can be general recognizing whole IgG and any fragments thereof, or can be specific recognizing only a single host species of primary antibody protein.  For this reason Rockland has developed affinity purified and highly cross adsorbed secondary antibodies. 

Antibodies may be directed against either the whole molecule IgG, designated as "(H&L)" for heavy and light chains, or the only an antibody fragment that for example could include the light chain, F(c) portion of IgG, or the F(ab')2 portion of IgG.

Pre-adsorbtion (also cross-adsorbtion) of the secondary antibody is used to eliminate reactivity to IgG from undesired host species or antibody fragments.    The degree of cross reactivity is determined by ELISA and is typically less than 1% of the desired signal.   The secondary antibody is cross adsorbed against serum antibody protein from another species or is adsorbed against a mixture of serum antibody protein from several species (i.e., Pre-adsorbed). These highly cross adsorbed antibodies show low levels of cross reactivity in multiple labeling experiments. Cross reactivity of Pre-adsorbed secondary antibodies is determined by ELISA or Western Blot Detection and is typically less than 1% or of the desired signal.

Many antibodies are offered as pre adsorbed against serum proteins from another species or are adsorbed against a mixture of serum proteins from several species (ie. designated "X to Ch,GP,Ham,Hs,Ms,Rb & Rt"). These highly cross adsorbed antibodies show extremely low levels of cross reactivity in multiple labeling experiments. The degree of cross reactivity is determined by ELISA and is typically less than 1% of the desired signal.

See a list of pre-adsorbed antibodies offered by Rockland

 


Related products

See all related substrate products from Rockland


Peroxidase enzyme substrates for ELISA 
Peroxidase enzyme substrates for WB and IHC
Blocking buffers
• Chemiluminescent FemtoMax™ Super Sensitive HRP Substrate FEMTOMAX-110
• NPP (50X) ELISA Alkaline Phosphatase Substrate NPP-10