Technical Data

I1904-84A2
Clone Type
Monoclonal
Host
Mouse
Source
Human
Isotype
IgG2a
Clone Number
F10-89-4
Grade
Highly Purified
Applications
FC
Crossreactivity
Hu
Shipping Temp
Blue Ice
Storage Temp
-20°C
Mouse Anti-IgG2a, Murine Negative Isotype Control (for rat tissue)
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.
Applications Suitable for use in Flow Cytometry (for Use With Rat Tissues Only) and Matched Negative Control Reagent. Other applications not tested.
Recommended Dilutions
Flow Cytometry: Neat. Use 10ul of the optimal dilution to label 10e6 cells in 100ul. Matched Negative Control Reagent: Assess the level of non-specific binding of mouse IgG2a monoclonal antibodies to rat cells/ tissue. Optimal dilutions to be determined by researcher.
Positive Control: Human leukocytes
Recommended Secondary Antibody
Goat anti-mouse IgG labeled with RPE (rat adsorbed) Goat anti-mouse IgG labeled with FITC (rat adsorbed)
Fusion Partners: Spleen cells from immunized BALB/c mice were fused with cells of the NSI mouse myeloma cell line.
Storage and Stability
May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Immunogen
Human T lymphocytes.
Form
Supplied as a liquid in PBS, pH 7.2, 1% BSA, 0.09% sodium azide.
Purity
Purified by Ion Exchange chromatography.
Specificity
Recognizes the human CD45 antigen. Species Crossreactivity: Negative on the following rat tissues-peripheral blood leucocytes, thymocytes, splenocytes and macrophages.

Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.

References
No references available
USBio References
No references available
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