This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometry and immunofluorescent analysis in human cells. The unconjugated antibody #4545 reacts with keratins 4, 5, 6, 8, 10, 13 and 18 from human, rat and monkey. CST expects that Pan-Keratin (C11) Mouse mAb (Alexa Fluor® 488 Conjugate) will also recognize the same keratins in these species.
Product Usage Information
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.
posted December 2010
Protocol Id: 221
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).
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
Protocol Id: 407
Specificity / Sensitivity
Pan-Keratin (C11) Mouse mAb (Alexa Fluor® 488 Conjugate) detects endogenous levels of total keratins 4, 5, 6, 8, 10, 13 and 18. The antibody does not cross-react with other keratins.
Human, Mouse, Rat, Monkey
Source / Purification
Monoclonal antibody is produced by immunizing animals with a cytoskeleton preparation from A431 cells. The antibody was conjugated to Alexa Fluor® 488 under optimal conditions with an F/P ratio of 2-6.
Keratins (cytokeratins) are intermediate filament proteins that are mainly expressed in epithelial cells. Keratin heterodimers composed of an acidic keratin (or type I keratin, keratins 9 to 23) and a basic keratin (or type II keratin, keratins 1 to 8) assemble to form filaments (1,2). Keratin isoforms demonstrate tissue- and differentiation-specific profiles that make them useful as research biomarkers (1). Research studies have shown that mutations in keratin genes are associated with skin disorders, liver and pancreatic diseases, and inflammatory intestinal diseases (3-6).
For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.