Revision 12
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, IHC-P, IF-IC, FC-FP

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

17, 19

SOURCE:

Rabbit

UniProt ID:

#P42574

Entrez-Gene Id:

836

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:400
Immunofluorescence (Immunocytochemistry) 1:400
Flow Cytometry (Fixed/Permeabilized) 1:800

Storage

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

Specificity / Sensitivity

Cleaved Caspase-3 (Asp175) Antibody detects endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. This antibody does not recognize full length caspase-3 or other cleaved caspases. This antibody detects non-specific caspase substrates by western blot. Non-specific labeling may be observed by immunofluorescence in specific sub-types of healthy cells in fixed-frozen tissues (e.g. pancreatic alpha-cells). Nuclear background may be observed in rat and monkey samples.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology

Bovine, Dog, Pig

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino-terminal residues adjacent to (Asp175) in human caspase-3.

Background

Caspase-3 (CPP-32, Apopain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (1). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires the aspartic acid residue at the P1 position (2).

  1. Fernandes-Alnemri, T. et al. (1994) J Biol Chem 269, 30761-4.
  2. Nicholson, D.W. et al. (1995) Nature 376, 37-43.

Species Reactivity

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

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

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.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 12
#9661

Cleaved Caspase-3 (Asp175) Antibody

Western Blotting Image 1: Cleaved Caspase-3 (Asp175) Antibody Expand Image
Western blot analysis of extracts from HeLa, NIH/3T3 and C6 cells untreated, staurosporine-treated (3hrs, 1 µM in vivo) or cytochrome c-treated (1hr, 0.25 mg/ml in vitro), using Caspase-3 Antibody #9662 (upper) or Cleaved Caspase-3 (Asp175) Antibody (lower).
Western Blotting Image 1: Cleaved Caspase-3 (Asp175) Antibody Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from Jurkat cells treated with Cytochrome C using Cleaved Caspase-3 (Asp175) Antibody #9661. The virtual lane view (left) shows two target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Immunoprecipitation Image 1: Cleaved Caspase-3 (Asp175) Antibody Expand Image
Immunoprecipitation of cleaved caspase-3 from Jurkat extracts treated with Etoposide #2200 (25 mM; 5 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Cleaved Caspase-3 (Asp175) Antibody. Western blot analysis was performed using Cleaved Caspase-3 (Asp175) Antibody. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Immunohistochemistry Image 1: Cleaved Caspase-3 (Asp175) Antibody Expand Image
Immunohistochemical analysis of paraffin-embedded human tonsil, showing cytoplasmic and perinuclear localization in apoptotic cells (low and high magnification), using Cleaved Caspase-3 (Asp175) Antibody.
Immunohistochemistry Image 2: Cleaved Caspase-3 (Asp175) Antibody Expand Image
Immunohistochemical analysis using Cleaved caspase-3 (Asp175) antibody on SignalSlide Cleaved Caspase-3 IHC controls #8104 (paraffin-embedded Jurkat cells, untreated (left) or etoposide-treated (right)).
Immunohistochemistry Image 3: Cleaved Caspase-3 (Asp175) Antibody Expand Image
Immunohistochemical analysis of paraffin-embedded mouse embryo, using Cleaved Caspase-3 (Asp175) Antibody preincubated with control peptide (left) or Cleaved Caspase-3 (Asp175) Blocking Peptide #1050 (right).
Immunofluorescence Image 1: Cleaved Caspase-3 (Asp175) Antibody Expand Image
Confocal immunofluorescent images of HT-29 cells, untreated (left) or Staurosporine #9953 treated (right), labeled with Cleaved Caspase-3 (Asp175) Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5® (fluorescent DNA dye).
Flow Cytometry Image 1: Cleaved Caspase-3 (Asp175) Antibody Expand Image
Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with etoposide #2200 (green), using Cleaved Caspase-3 (Asp175) Antibody compared to a nonspecific negative control antibody (red).