Choosing an antibody

How to navigate the Abcam catalog and find the antibodies most suitable for your experiments

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There is often more than one antibody available for any given target. To narrow the range of choice, several aspects of the experiment need to be considered:

  1. Type of assay or application
  2. Nature of sample
  3. Species of the sample
  4. Species of the antibody host
  5. Labelling and detection of the antibody

The Abcam website has a useful search function. Entering the name of the protein or other target in the search box will generate a list that can be filtered by product type, target, applications tested, species reactivity, host species, clonality, and conjugation.

Application

Antibody datasheets list the applications that have been tested and found to work. If an application is not listed, this does not mean that the antibody is not suitable. It simply means that it has not been tested and it is unknown how the antibody will perform. When an application has been tested and found not to work, this will be noted on the datasheet.

Nature of sample

The nature of the sample will dictate which antibody will work best. At least two aspects need to be considered:

1. The region of the protein one wishes to detect. Antibodies are generated by immunization of host animals with a variety of immunogenic substances including full-length proteins, protein fragments, peptides, whole organisms (for example bacteria), or cells. The immunogen is generally described on the datasheet (though in many cases an exact description of the immunogen is not available for proprietary reasons). If trying to detect a protein fragment or a specific isoform or region of the full-length protein, one needs to be sure to choose an antibody that is raised against an immunogen that is identical to or contained within the fragment or region. If trying to detect a cell surface protein on live cells by FACS, one needs to choose an antibody that is raised against an extracellular domain of the protein.

2. Processing of the sample. Some antibodies require samples to be processed or treated in a specific manner. For instance, many antibodies will only recognize proteins that have been reduced and denatured, presumably because this reveals epitopes that would otherwise be obscured by secondary and tertiary folding of the proteins. On the other hand, some antibodies will only recognize epitopes on proteins in their native, folded state. (For Abcam antibodies to be used for western blotting, samples should be reduced and denatured unless otherwise noted on the datasheet). When searching for an antibody for immunohistochemistry, it should be noted that some antibodies are only appropriate for unfixed frozen tissue, and many others are incapable of binding to their targets in formalin-fixed, paraffin-embedded tissues without an antigen retrieval step that reverses the cross-links introduced by formalin fixation. These restrictions on use will be noted in the applications section of datasheets.

Species of sample

An antibody should be chosen that has been raised against the same species one is studying. The antibody may react with the same target protein from other species sharing sufficient amino acid sequence homology. If the sample is not from one of the species listed, this does not mean that the antibody will not detect the protein, but rather that the species has not been tested and we are reluctant to comment on its suitability. A prediction of cross-reactivity can be made based on sequence similarity: Abcam is now proud to have Expasy and NCBI BLAST links on the datasheets to compare amino acid sequence homology among different species.

Choosing the species of primary antibody host

In general, the species of the host animal in which an antibody was raised is important when using a conjugated secondary antibody to detect an unconjugated primary. For immunohistochemistry, the primary antibody should be raised in a species as phylogenetically different as possible from the species of the sample. This is to avoid potential cross-reactivity of the secondary anti-immunoglobulin antibody with endogenous immunoglobulins in the sample. For instance, a primary antibody used to detect a protein in a sample from a mouse should not be raised in mouse or rat. A primary antibody raised in rabbit will be a more appropriate choice, followed by an anti-rabbit IgG secondary antibody conjugated to a detection molecule (enzyme, fluorochrome, biotin, etc.). The issue can be avoided if a conjugated primary antibody is available. For other techniques using samples that do not contain endogenous immunoglobulin, the choice of host species is less critical. An example would be western blotting of a cell lysate that is not expected to contain IgG. However, tissue lysates and tissue culture supernatants that contain serum will contain immunoglobulins. IgG will appear in western blots of reduced, denatured samples as bands at 50 and 25 kDa corresponding to the heavy and light chains of the IgG molecule.

Choosing a secondary antibody

Secondary antibodies should be raised against the same species as the primary antibody you are using. For example, if your primary is a mouse monoclonal, you will require an anti-mouse secondary.  We recommend you check the datasheet of the secondary antibody to ensure it is tested in the application you will be using.  Abcam provide a wide range of secondary antibodies conjugated to a range of fluorochromes and chromogens.

  1. Check the bottom of the datasheet of the primary antibody you are using -   there will be a list of suitable secondary antibodies
  2. To find your own secondary, select this product type using our navigation bar, or by using the links at the center of the homepage. Then choose the species of your primary antibody. This will then give you a range of secondary antibodies for use with that species of primary.  

Choosing antibodies for dual staining

Double immunostaining of cell cultures or tissue sections using unconjugated primary antibodies requires that those antibodies are raised in different species and that the secondary antibodies recognize one of those species exclusively. Datasheets for secondary anti-Ig antibodies will state if they have been cross-adsorbed against immunoglobulins from other species to remove those reactivities.

Fluorochrome and chromogen labels

Labels are conjugated (joined) to antibodies in order to visualize the binding of the antibody. The choice of label depends on several parameters:

1. Detection method: fluorescence or colored precipitate. Fluorescent labels emit light in the visual range when excited by light of a specific wavelength. There are several available, all with their own excitation and emission characteristics:

Fluorochrome

Color

Excitation
(nm)

Emission
(nm)

Molecular
Weight
(daltons)

Aminomethylcoumarin (AMCA)

Blue

353

440

410

Fluorescein (FITC)

Green

495

528

390

Fluorescein (DTAF)

Green

495

528

530

Rhodamine (TRITC)

Red

550

570

444

Texas Redtm

Red

596

620

625

Cy2tm

Green

489

505

897

Cy3tm

Orange

552

565

949

Cy3.5tm

Scarlet

581

596

1,286

Cy5tm

Far-Red

650

667

975

Cy5.5tm

Near Infra-Red

678

703

1,312

Cy7tm

Near Infra-Red

743

767

1,001

R-Phycoerythrin (RPE)

Red

488

575

240,000

B-Phycoerythrin (BPE)

Red

545

575

240,000

C-Phycocyanin

Red

618

640

110,000

R-Phycocyanin

Red

615

620

110,000

When combined with the appropriate substrate the enzymatic labels HRP and AP form a colored precipitate:

Horseradish peroxidase (HRP)

Alkaline phosphatase (AP)

AEC (red)

Fast red (pink)

DAB (brown)

INT (yellow / brown)

 

NBT (brown / purple)

 

New Fuchsin (red)

 

TNBT (purple)

 

Vega red (pink)

2. Available mounting media (immunohistochemistry only). AEC, Fast Red, INT or any other aqueous chromogen are alcohol soluble and require an aqueous based mounting medium. The others mentioned above are organic, so are best mounted in organic mounting media in order to take advantage of the better refractive index.

Fluorescent labels require aqueous mounting media. Phycobiliproteins (phycocyanin / phycoerythrin) require aqueous mounting media with no added glycerol, since this has a quenching effect.

3. Biotinylated antibodies are useful for amplification of signal when followed by an avidin-biotin-enzyme or fluorochrome complex (commonly abbreviated as “ABC” reagent), or avidin or streptavidin conjugated to an enzyme or fluorochrome.

Back to Antibody Guide index

Next: Antibody dilutions and titer