Description
The G15-337 monoclonal antibody specifically recognizes rat IgG2b. It does not react with other Ig isotypes. A suspension of pooled rat Ig was used as the source of immunogen.
Format
- FormatFITC
- Excitation SourceBlue 488 nm
- Excitation Max494nm
- Emission Max520nm
FITC, fluorescein isothiocyanate, is a fluorochrome with a molecular weight of 389 Da. FITC is sensitive to pH changes and photobleaching. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor® 488 cannot be used simultaneously. FITC is relatively dim and should be reserved for highly expressed markers whenever possible.
Other Formats→
- BB700
- BUV395
- BUV496
- BUV615
- BUV661
- BV421
- BV480
- BV510
- BV605
- BV650
- BV711
- BV750
- BV786
- Biotin
- Purified
Suggested Companion Products
FITC Mouse IgG2b, κ Isotype Control RUO 0.1mgCat No: 555057
Stain Buffer (FBS) RUO 500mLCat No: 554656
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) 2.4G2RUO 0.1mgCat No: 553141
Purified Mouse Anti-Rat IgG2b G15-337RUO 0.5mgCat No: 553882
BD Cytofix/Cytoperm™ Fixation/Permeablization Kit RUO 250TestsCat No: 554714
Stain Buffer (BSA) RUO 500mLCat No: 554657
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) 2.4G2RUO 0.5mgCat No: 553142
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Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light.Do not freeze.The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
G15-337 antibody is effective for detection of cell-surface or intracellular Ig by immunofluorescent staining with flow cytometric analysis. FITC-conjugated G15-337 mAb (Cat. No. 553884) may be used as a primary or secondary reagent in immunofluorescent staining.
IMMUNOFLUORESCENT STAINING OF INTRACELLULAR
IMMUNOGLOBULIN (Ig) PROTOCOL
1. Prepare a single-cell suspension and determine cell number.
2. Suspend cells in staining buffer (PBS + 2% FBS + 0.1% Sodium Azide) at 2 x 10e7 cells/ml and transfer to U-bottom microwell plates in 50µl/well for immunofluorescent staining.
Note: The BD Pharmingen™ Stain Buffer with FBS (Cat. No. 554656) is effective for use as a staining buffer in this protocol.
3. Block Fcγ receptors by adding 0.2 µg of purified 2.4G2 antibody (Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2, Cat. No. 553141/553142),in 50 µl of staining buffer to each well.
4. Incubate 5 minutes on ice.
5. Add 200 µl of staining buffer/well and resuspend cells. Centrifuge at 250 x g for 5 minutes and aspirate supernatant.
6. Block surface Ig with purified G15-337 mAb (Cat. No. 553882) by adding 1.0 µg per sample in 50 µl of staining buffer/well.
Note: Surface markers may be stained during this step as described in the "Multicolor Flow Cytometry" protocols at our website: http://www.bdbiosciences.com/us/s/resources
7. Incubate 15 minutes on ice.
8. Wash 2x as described in Step 5.
9. Resuspend cells in 100 µl of BD Cytofix/Cytoperm™ intracellular staining buffer (see BD Cytofix/Cytoperm™ Kit, Cat. No. 554714) per well.
10. Incubate 30 minutes at room temperature.
11. Wash 2x with 200 µl of 1x Perm/Wash buffer (provided in the BD Cytofix/Cytoperm Kit) per well. Centrifuge at 250 x g for 5 minutes and aspirate supernatant between washes.
12. Stain intracellular Ig by adding ≤ 1 µg of FITC-conjugated G15-337 mAb (Cat. No. 553884) in 50 µl of 1 x Perm/Wash buffer/well.
Note: Other antibodies recommended for staining of intracellular markers may be added during this step as described in Step 12.
13. Incubate for 30 minutes at room temperature.
14. Wash 2x as described in Step 11.
15. Resuspend and transfer samples in 100 µl of staining buffer to tubes appropriate for analysis with a flow cytometer. Bring volume in each tube to 400 µl with staining buffer.
16. Analyze samples on a flow cytometer.
