PathScan® Apoptosis and Proliferation Multiplex IF Kit (#7851) Datasheet with Images Cell Signaling Technology

For Research Use Only. Not for Use in Diagnostic Procedures.

Products Included	No.	Volume	Applicaton	Dilution	Reactivity	Homology†
Primary Cocktail	7847	100 µl	Immunofluorescence (Immunocytochemistry), Immunofluorescence (Paraffin)‡	1:100	Human	Monkey
Detection Cocktail	7843	100 µl	Immunofluorescence (Immunocytochemistry), Immunofluorescence (Paraffin)‡	1:100	N/A	N/A

†Species predicted to react based on 100% sequence homology.

‡Immunofluorescence (Paraffin) protocol recommended unmasking buffer: Citrate

Kit Analytes	Detection Dye	Ex_(max) (nm)	Em_(max) (nm)
Phospho-Histone H3 (Ser10)	Alexa Fluor^® 488	495	519
Cleaved-PARP (Asp214)	Alexa Fluor^® 647	650	665
α-Tubulin	Alexa Fluor^® 555	555	565

Applications:

IF-P

UniProt ID:

#P68431, #P68363, #P09874

Entrez-Gene Id:

8350, 10376, 142

Product Information

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 μg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

α-Tubulin antibody detects endogenous levels of total α-tubulin protein. Phospho-Histone H3 (Ser10) antibody detects endogenous levels of histone H3 only when phosphorylated at Ser10. This antibody does not cross-react with other phosphorylated or acetylated histones. Cleaved-PARP (Asp214) detects endogenous levels of the large fragment (89 kDa) of human PARP1 protein produced by caspase cleavage. This antibody does not recognize full length PARP1 or other PARP isoforms.

Species Reactivity:

Human

Source / Purification

Monoclonal antibodies were produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3, a synthetic peptide corresponding to residues surrounding Asp213 of human PARP, or with full-length chicken α-tubulin purified from brain extracts.

Product Description

The PathScan^® Apoptosis and Proliferation Multiplex IF kit offers a novel method to simultaneously monitor mitotic index and programmed cell death using manual immunofluorescence microscopy, or automated imaging and laser scanning high content platforms. This kit contains a cocktail of three high quality primary antibodies targeted against α-tubulin, phospho-histone H3 (Ser10), and cleaved PARP (Asp214), as well as a detection cocktail utilizing the Alexa Fluor^® series of fluorescent dyes. Antibody and dye pairings have been pre-optimized, and each kit contains enough reagents for 100 assays (based on a working volume of 100 μL/test).

Background

Apoptosis is a regulated physiological process leading to cell death. Initiator caspases cleave and activate downstream effector caspases that in turn cleave cytoskeletal and nuclear proteins like PARP, α-fodrin, DFF, and lamin A, which induce apoptosis (1). Cleavage of the nuclear poly (ADP-ribose) polymerase PARP occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (2,3). PARP helps to maintain cell viability by playing key roles in many cellular processes, including DNA replication, repair, and recombination. Cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (4). Cell proliferation can be measured by studying the phosphorylation state of histone H3 and microtubule assembly during mitosis. Histone H3 is one of four distinct core histone proteins found in the nucleosome. Phosphorylation of histone H3 at Ser10, Ser28, and Thr11 is tightly correlated with chromosome condensation during both mitosis and meiosis (5-7). Heterodimers composed of α-tubulin and β-tubulin form globular tubulin subunits common to all eukaryotic cells. Tubulin polymers known as microtubules are fundamental cytosolic fibers important in mediating cellular movement, including meiotic/mitotic chromosome alignment, cytoplasmic membrane vesicle transport, and nerve-cell axon migration (8).

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Applications Key

IF-P: Immunofluorescence (Paraffin)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

PathScan is a registered trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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