Isotype Controls:Isotype controls are a type of negative control designed to measure the level of non-specific background signal caused by primary antibodies, based upon the tissue type of the sample. Non-specific binding of primary antibodies are typically caused by the following reasons:
Non-specific Fc receptor binding to cells
Non-specific antibody interactions with cellular proteins or fluorochromes
Non-specific binding of the antibody or fluorochromes to cellular components
Cell autofluorescence
Usually, the background signal is the result of immunoglobulins binding non-specifically to Fc receptors present on the cell surface.
For example, antibodies raised in mice, particularly those of the IgG2a isotype, will bind strongly to some human leukocytes regardless of the test antibody specificity.
In this case you would need a mouse IgG2a isotype control for use with human cells or tissues.
Isotype controls are most commonly used in flow cytometry experiments, but may also be used in immunohistochemistry.
Selecting an Isotype Control:Typically, an isotype control is matched to the host species and isotype of your specific primary antibody, the heavy chain (IgA, IgG, IgD, IgE, or IgM), subclass and light chain (kappa, lambda) class of the primary antibody; the same fluorochrome (PE, APC, etc.), and have the same F:P ratio.
F:P is a measurement of how many fluorescent molecules are present on each antibody.
When using directly labeled primary antibodies, it is also necessary to make sure that the isotype control is conjugated to the same fluorochrome or label as the test antibody.
It is not sufficient to use one with a spectrally similar fluorochrome.
Non-specific Binding:To avoid non-specific binding, you also need to block Fc receptors on cell types, such as spleen cells, with FcR blocking reagents.
Isotype controls should be used at the same concentration/dilution as the primary antibody.
Isotype controls are mainly used to determine if the staining is real.
Apart from isotype controls, unstained cells should always be included in the experimental set-up to monitor autofluorescence.
Applications for Isotype Controls:Although isotype controls are mainly used in flow cytometry, they can be used as standard blocking agents and protein coating agents for other applications including immunofluorescence, immunocytochemistry, western blotting, and ELISA.
Isotype control antibodies are used to estimate the non-specific binding of target primary antibodies due to Fc receptor binding or other protein-protein interactions.
An isotype control antibody should have the same immunoglobulin type as the test antibody.
Rabbit IgG was conjugated to Alexa Fluor® 647 fluorescent dye under optimal conditions, and tested in-house for direct flow cytometric analysis of human and mouse cells.
Alexa Fluor® dye is maximally excited by red light (e.g.
633nm He-Ne laser).
Antibody conjugated of the Alexa Fluor® 647 dye produce far-red-fluorescence emission with a peak at 655nm.
Directions on Use:Important! This control antibody must be diluted to the same concentration (not dilution) as the specific antibody in analysis.
Higher background fluorescence may result if excessive amounts of rabbit IgG isotype control are used.
Do not use this antibody at 1:50 dilution in the same way as recommended for other Alexa Fluor® conjugated antibodies.
Applications:Suitable for use in Flow Cytometry.
Other applications not tested.
Recommended Dilutions:Optimal dilutions to be determined by the researcher.
Storage and Stability:May be stored at 4°C before opening.
DO NOT FREEZE! Stable at 4°C as an undiluted liquid.
Dilute only prior to immediate use.
Stable for 12 months after receipt at 4°C.
Freezing Alexa Fluor® conjugates will result in a substantial loss of activity.
Alexa Fluor® conjugates are sensitive to light.
仕様
Size：100ul
Host：rabbit
Grade：Affinity Purified
Purity：Purified by affinity chromotography
Form：Supplied as a liquid in PBS, pH 7.2, 2mg/ml BSA, <0.09% sodium azide. Labeled with Alexa Fluor® 647 (F/P ratio of 2-5)
