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To Purchase # 14411S
|14411S||100 µl (100 immunocytochemical stainings)||$109.00.0|
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- Additional protein information
- Analytical tools
Mouse (G3A1) mAb IgG1 Isotype Control (Pacific Blue™ Conjugate) #14411
Gallery: Mouse (G3A1) mAb IgG1 Isotype Control (Pacific Blue™ Conjugate) #14411
Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 16% Formaldehyde (methanol free).
- 100% methanol.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
- Fix for 15 min at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
- Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
- Incubate 30 min on ice.
- Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
- Aliquot desired number of cells into tubes or wells.
- Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 1 hr at room temperature. Protect from light.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).
E. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted July 2009
revised June 2017
Note: This control antibody must be diluted to the same concentration (not dilution) as the specific antibody in analysis. See Directions on Use.Directions for Use: Important! This control antibody must be diluted to the same concentration (not dilution) as the specific antibody used for 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 Pacific Blue conjugated antibodies.
Important! Dilute this control antibody to the same concentration (not dilution) as the specific antibody used for analysis. Higher background may result if excessive amounts of mouse IgG1 isotype control is used.Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Protect from light. Do not freeze.
Mouse (G3A1) mAb IgG1 Isotype Control (Pacific Blue™) is not directed against any known antigen. It functions as an isotype control for mouse IgG1 monoclonal antibodies.
This Cell Signaling Technology antibody is conjugated to Pacific Blue™ fluorescent dye and tested in-house for flow cytometry analysis in human cells.
Isotype control antibodies are used to estimate the nonspecific 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 and be used at the same concentration as the test antibody.
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Pacific Blue is a trademark of Molecular Probes, Inc.