Biolegend, 628604, Alexa Fluor® 647 anti-Cytokeratin (pan reactive) Antibody, 100μg

Cytokeratins are intermediate filament proteins that are widely expressed in many tissues. These proteins are vital components of the cytoskeleton and interact with a number of other proteins. Cytokeratins are usually found in the cytoplasm,although some cytokeratins can be found in the nucleus and the nucleolus.
100μg
Verified Reactivity: Human
Antibody Type: Monoclonal
Host Species: Mouse
Immunogen: Keratin enriched fraction from human epidermoid carcinoma.
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation: The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 647 under optimal conditions.
Concentration: 0.5 mg/ml
Storage & Handling: The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application: ICC - Quality tested ICFC - Verified SB - Reported in the literature, not verified in house
Recommended Usage: Each lot of this antibody is quality control tested by immunocytochemistry. For immunocytochemistry, a concentration range of 1.0 - 5.0 μg/ml is recommended. For flow cytometric staining, the suggested use of this reagent is ≤ 0.125 µg per million cells in 100 µL volume. It is recommended that the reagent be titrated for optimal performance for each application. * Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633 nm / 635 nm. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Excitation Laser: Red Laser (633 nm)
Application Notes: The C-11 monoclonal antibody reacts with a conserved epitope of human cytokeratin 4, 5, 6, 8, 10, 13, and 18. This antibody has been shown to be useful for Western blotting and has also been reported to be useful for immunoprecipitation, immunohistochemistry (paraffin sections), immunocytochemistry, and spatial biology (IBEX)7,8.
Additional Product Notes: Iterative Bleaching Extended multi-pleXity (IBEX) is a fluorescent imaging technique capable of highly-multiplexed spatial analysis. The method relies on cyclical bleaching of panels of fluorescent antibodies in order to image and analyze many markers over multiple cycles of staining, imaging, and, bleaching. It is a community-developed open-access method developed by the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH).
Application References(PubMed link indicates BioLegend citation): Kovarik J. 1988. Int. J. Cancer Suppl. 3:50. Bartek J, et al. 1991. J. Pathol. 164:215. Chernyavsky A I, 2007. J. Biol. Chem. doi:10.1074/jbc.M611365200. PubMed Comito G, et al. 2012. Cancer Lett. 324:31. PubMed Vojt-òsek B, et al. 1989. Folia Biol (Praha). 35:373. (reactivity with rat, dog, sheep, pig, cow) Dekaney CM, et al. 2005. Gastroenterology 129:1567. (ICC) Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations: Roy S, et al. 2019. J Cell Biochem. 120:4804. PubMed Hendricks A, et al. 2021. Front Oncol. 11:646885. PubMed Hendricks A, et al. 2020. Cancers (Basel). 12:00. PubMed Vishnoi M, et al. 2019. Mol Oncol. 13(9): 1913. PubMed
RRID: AB_2563652 (BioLegend Cat. No. 628604)
Structure: Intermediate filament protein, contains three coiled-coil domains. Cytokeratins exist as heterotetramers composed of two type I and two type II keratin subunits
Distribution: Cytokeratins are widely expressed proteins usually found in the cytoplasm although some cytokeratins can also be found in the nucleus and the nucleolus
Function: Intermediate filament protein involved with the cytoskeleton.
Interaction: Most cytokeratins interact with a number of other proteins such as 14-3-3, EGF receptor, and many others
Biology Area: Cell Biology, Cell Motility/Cytoskeleton/Structure, Neuroscience, Neuroscience Cell Markers
Molecular Family: Intermediate Filaments
Gene ID: 3875
UniProt: View information about Cytokeratin pan reactive on UniProt.org
Clone: C-11
Regulatory Status: RUO
Other Names: Cytokeratin, Keratin
Isotype: Mouse IgG1, κ
Q: If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?
A: It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.
Q: Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?
A: Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.
Q: Are other fluorophores compatible with IBEX?
A: Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.
Q: The same antibody works in one tissue type but not another. What is happening?
A: Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.
Q: How can I be sure the staining I’m seeing in my tissue is real?
A: In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.