Alexa Fluor® 647 Rat IgG2a, κ Isotype Control
Clone R35-95 (RUO)
- Brand BD Pharmingen™
- Vol. Per Test 5 µl
- Isotype Rat LOU, also known as Louvain, LOU/C, LOU/M IgG2a, κ
Isotype control, Intracellular staining (flow cytometry) (Routinely Tested)
- Immunogen Mouse Pooled Immunoglobulin
- Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
The R35-95 hybridoma was generated by hybridization of Y3 myeloma cells with spleen cells from LOU rats immunized with mouse immunoglobulins. The R35-95 hybridoma produces rat IgG2a, κ immunoglobulin that has no measurable reactivity with mouse immunoglobulins. The R35-95 immunoglobulin was selected as an isotype control following screening for low background binding on a variety of mouse and human tissues.
This antibody is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
Alexa Fluor® 647 conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of APC. However, APC tends to be brighter while Alexa Fluor® 647 is more optimal for intracellular applications. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.
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Preparation and Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
An isotype control should be used at the same concentration as the antibody of interest (e.g., ≤ 1 µg/million cells for flow cytometry).
Important Note: This pre-titered antibody solution does not contain a cell permeabilization agent. It is necessary to include a cell permeabilization agent when using the pre-titered antibody solution to stain fixed and permeabilized cells with Cytofix/Cytoperm Kit(Cat. No. 554714). BD Perm/Wash™ Buffer (Cat. No 554723) contains the permeabilization agent saponin and is useful for this purpose as described in the USAGE section below.
USAGE for Intracellular Staining
Resuspend 1 x 10e6 fixed and permeabilized cells in 5 µl of the pre-titered antibody solution and 45 µl of 1X Perm/Wash Buffer (Cat. No 554723).
Incubate the cell suspension for 15 minutes (at RT or 4°C).
Wash twice in 100 µl of 1X Perm/Wash Buffer (Cat. No 554723).