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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. Primary antibodies are typically produced in mouse, rabbit, goat or chicken host species; therefore, anti-mouse IgG, anti-rabbit IgG, anti-goat IgG or anti-chicken polyclonal antibodies are used. Identifying the optimal secondary antibody is normally done based on the nature of the primary antibody and the detection assay. Secondary antibodies are used throughout various types of assays, including ELISA or Western Blot, the secondary antibody type is selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred.


•  Chicken Secondary Antibodies  •  Mouse Secondary Antibodies  
•  Goat Secondary Antibodies   •  Rabbit Secondary Antibodies 

•  Monkey Secondary Antibodies

 

 

 

 

 
 
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Conjugated Secondary Antibodies

 

Horse Radish Peroxidase (HRP) Conjugated Secondary Antibody is the most popular conjugated antibody. It can be used in Western Blot Analysis (immunoblotting), ELISA and Immunohistochemistry. Beside the HRP conjugated secondary antibodies, Rockland further produces secondary antibodies that are FITC conjugated, as well as conjugates to Rhodamine, Texas Red and Phycoerythrin (PE) and dyes for multiplex platforms like the LI COR Odyssey Imaging System or Molecular Devices multi-mode readers. These dyes consist of Atto Dyes and DyLight™ Dyes.

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Cross Adsorbed Secondary Antibodies

 

Secondary antibodies can be specific (e.g., recognize only one host species of primary antibody protein) or can be general (e.g., recognize whole IgG and any fragments thereof). For this reason Rockland has developed highly cross adsorbed secondary antibodies. 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 (e.g. 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.



F(ab’)2 Fragment Biotinylated Secondary Antibodies

 

F(ab')2 fragment biotinylated 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. F(ab')2 fragment biotinylated antibodies eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells. If 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 anti biotin antibody binding through Fc receptors present on cells. F(ab')2 fragment conjugated secondary antibodies are ideal for Flow Cytometry, Immunohistochemistry and Immunofluorescence.

 

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Phospho Related Antibodies

 

 

 

TrueBlot IP/Western Blot Mouse Rabbit Goat

 

 

 

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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  

  

 

 

Clear Results, Enhanced Accuracy

 

Rockland offers a number of TrueBlot® products to assist in the accurate detection and isolation of secondary antibodies utilized in a variety of assays. TrueBlot® products help eliminate heavy/light chain blotting and contamination from immunoprecipitation that can derail many projects. 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.



 

Rockland Secondary Antibody Conjugates

 

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.

  


Immunofluorescence Microscopy

 

Secondary, or indirect, immunofluorescence uses two antibodies; a primary antibody which recognizes the target biomolecule and binds to it and a secondary antibody conjugated to a fluorescent dye, which recognizes and binds to the primary antibody and indirectly localizes the target for detection by the microscope. While this protocol is more complex than the direct method, it is more flexible with regard to experimental design, results in greater signal detection through amplification and is easier in that secondary antibody conjugates are commercially available. Visit our Immunofluorescence Microscopy page for more information.




Immunohistochemistry Studies

 

Rockland's Immunohistochemistry Studies (IHC) provide confidential high-quality target localization data through its extensive services. Researchers are presented with the option of outsourcing their entire IHC studies, bypassing the difficulties of sourcing and characterizing the appropriate target-specific antibodies or difficult-to-find validated tissues and the complex immunostain interpretation thereof. Commonly requested standardized IHC services are offered as Service Packets; however, Rockland’s custom design of your required study plan will address your particular biological questions. All studies are conducted under R&D conditions, are confidential and the results are proprietary to the customer. Visit our Immunohistochemistry Studies page for more information.




Choosing Secondary Antibodies

 

Oftentimes secondary antibodies recognize only one host species of primary antibody or alternatively recognize the entire IgG and any fragments thereof. Choosing the appropriate secondary probe or sufficiently designing the experimental system can overcome this disparity. When choosing a new antibody, keep the following considerations in mind:

  1. Determine the host species of the primary antibody and select an appropriate host species for the secondary antibody.
  2. Consider cross-reactivity or specificity issues of the secondary antibody as well as the detection or purification method.
  3. Finally, consider requirements of the supplied secondary and the supplied state of the reagent.  
            

The Rockland Advantage

 

Protect your experiments with Rockland secondary antibodies. Compromise elsewhere. All Secondary Antibodies are backed by our RockSolid Antibody Guarantee.

 

 

Secondary Antibody Applications

 

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