Jackson ImmunoResearch uses the Biuret assay to determine protein concentration.
If protein concentration is obtained by using the mass extinction coefficient of 1.4 for a 1 mg/ml solution at OD280, the result will be 10-15% lower than the concentration obtained by the Biuret assay.

Jackson ImmunoResearch does not determine molecular weights by analytical methods. An approximate molecular weight of 160, 110 and 50 kDa can be used for whole IgG, F(ab’)2 and Fab fragment antibodies, respectively. To obtain an estimated molecular weight of any other product of interest, please contact Technical Service at help@jacksonimmuno.com.

For purchase of OEM products that have custom specifications (e.g. custom adsorptions, conjugations), please contact Technical Service at technical@stratech.co.uk. Jackson ImmunoResearch does not provide polyclonal or monoclonal antibody development or production services.

Jackson ImmunoResearch does not assay products for endotoxin content. For applications requiring low endotoxin consider using an endotoxin removal column, available from Thermo Fisher, Charles River Laboratories, and other suppliers.

For products that are sold by weight (mg), Jackson ImmunoResearch fills vials with slightly more product that the nominal mg amount (“Size” on the spec sheet). To calculate the actual product amount in a vial, multiply the protein concentration indicated on the spec sheet by the volume of dH2O needed to rehydrate the lyophilized pellet.

Background may be caused by the primary antibody. Perform a control experiment excluding the primary antibody to isolate the secondary antibody as the cause of background.
Improper blocking of the tissues or cells.
Cross-reactivity of the labeled secondary antibodies with endogenous immunoglobulins on the tissues or cells.
Inadequate washing.
Reactivity of the labeled secondary antibody with immunoglobulins in the diluent.
Not diluting the secondary antibodies far enough.

There is insufficient antigen present and an amplification protocol may be needed.
Change from a more sensitive detection method to a less sensitive detection method.
The primary antibody is diluted too far.
Enzyme activity is inhibited.
The secondary antibody does not recognize certain primary antibodies well.
The secondary antibody cross-reacts with immunoglobulins in the diluent.

The antibodies can be diluted in buffers such as PBS or TBS. A detergent such as Tween 20 (0.05% v/v) can be included to reduce non-specific binding. We recommend making the working dilution fresh on the day of use. Carrier proteins such as BSA or normal serum are not necessary, and in some cases can increase background staining due to protein-protein interactions.
Read more about diluents in our guide to blocking, controls and diluents.

For most applications, we recommend blocking with normal serum (5% v/v) from the same species as the host of the secondary antibody. Other commonly used reagents such as BSA and non-fat dry milk are suitable for some applications, but may increase background if the secondary antibody is directed against goat, sheep, or horse.
Read more about blocking in our guide to blocking, controls and diluents.

Incubation times of 30-60 minutes are recommended for surface interactions such as ELISA or Western blotting. For staining tissue, incubation times should be increased as necessary to allow for tissue penetration. Total wash time should be approximately equal to incubation time to allow unreacted antibody to diffuse out.

Immunoglobulin classes (IgA, IgD, IgE, IgG, IgM in mammals) are defined by the structure of their heavy chains. Minor differences on heavy chains are found between subclasses of each class. Jackson ImmunoResearch offers polyclonal antibodies that are specific for mouse subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3. We also have antibodies that are specific for camelid subclasses IgG 2+3. We currently do not have subclass-specific antibodies against other species.
Download Immunoglobulin Structure and Function Poster.

These products are the result of proteolytic digestion of IgG. Digestion with papain results in Fab and Fc fragments, and digestion with pepsin yields F(ab’)2 fragments.

Isotypes are distinct forms of antibody heavy and light chains, and may refer to class or subclass. Light chain isotypes are kappa and lambda, and heavy chain isotypes are alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), mu (IgM). The heavy chains may be further differentiated as subclasses, e.g. gamma 1 (IgG1).

Isotypes are distinct forms of antibody heavy and light chains, and may refer to class or subclass. Light chain isotypes are kappa and lambda, and heavy chain isotypes are alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), mu (IgM). The heavy chains may be further differentiated as subclasses e.g. gamma 1 (IgG1).
An isotype control is a negative control that verifies specific binding of either a primary or secondary antibody. The isotype control is the same class or subclass as the experimental antibody, but is not specific for the antigenic target. Isotype controls may be purified IgG from normal serum of the species host of the experimental antibody; or in the case of monoclonal antibodies may be monoclonals of the same subclass, directed against an irrelevant antigen. Isotype controls for unlabeled antibodies are unlabeled, and controls for labeled antibodies are labeled with the same reporter molecule (fluorophore, enzyme, or biotin) as the experimental antibody.