Revision 6

#98134

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

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

Applications:
W, W-S, IP, IF-IC, FC-FP

Reactivity:
H

Sensitivity:
Endogenous

MW (kDa):
18, 41, 43

Source/Isotype:
Rabbit IgG

UniProt ID:
#Q14790

Entrez-Gene Id:
841

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:50 - 1:250
Immunoprecipitation 1:200
Immunofluorescence (Immunocytochemistry) 1:400
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600

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.

For a carrier free (BSA and azide free) version of this product see product #62901.

Specificity/Sensitivity

Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit Monoclonal Antibody recognizes endogenous levels of caspase-8 protein only when cleaved at Asp374. This antibody will detect cleavage products containing the pro-domain with the p18 subunit (p43/p41) as well as the p18 subunit alone.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp374 of human caspase-8 protein.

Background

Apoptosis induced through the CD95 receptor (Fas/APO-1) and tumor necrosis factor receptor 1 (TNFR1) activates caspase-8 and leads to the release of the caspase-8 active fragments, p18 and p10 (1-3). Activated caspase-8 cleaves and activates downstream effector caspases such as caspase-1, -3, -6, and -7. Caspase-3 ultimately elicits the morphological hallmarks of apoptosis, including DNA fragmentation and cell shrinkage.

In addition to functioning as a key initiator caspase for extrinsic apoptosis, more recent studies have identified caspase-8 as a regulator of inflammatory necrotic cell death pathways such as necroptosis and pyroptosis (4,5). Activation of caspase-8 leads to cleavage of RIPK1 and RIPK3 to inhibit necroptosis (6,7). As a result, caspase-8 deficiency in mice, which is embryonic lethal, can be rescued by deletion of the necroptosis proteins RIPK3 or MLKL (8-11). Additionally, in some circumstances, caspase-8 is recruited to the inflammasome to trigger pyroptosis (12,13). Studies have also found that expression of an enzymatically inactive form of caspase-8 (C362S) causes embryonic lethality by inducing necroptosis and pyroptosis (14).

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 BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

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

Cross-Reactivity Key

H: Human

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

使用に関する制限

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Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 6

Cell Signaling Technologyロゴ
Western blot analysis of extracts from HCT 116 and CRISPR/Cas9 caspase-8 knockout (KO) HCT 116 cells, untreated (-) or treated with Staurosporine #9953 (1 μM, 4 hr; +), using Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit mAb (upper), Caspase-8 (1C12) Mouse mAb #9746 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western Blotting Image 1: Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit Monoclonal Antibody
Western blot analysis of extracts from THP-1 cells, untreated (-) or treated (+) as indicated with: Cycloheximide #2112 (10 μg/mL, 18 hr) followed by Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 4 hr) using Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit mAb (upper), Caspase-8 (1C12) Mouse mAb #9746 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western Blotting Image 2: Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit Monoclonal Antibody
Western blot analysis of extracts from Jurkat cells, untreated (-) or treated with Etoposide #2200 (1 μM, 18 hr; +), using Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit mAb (upper), Caspase-8 (1C12) Mouse mAb #9746 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western Blotting Image 3: Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 6

Cell Signaling Technologyロゴ
Simple Western™ analysis of Jurkat cell lysates (1 mg/mL) treated with Cytochrome C (0.25mg/mL, 45 min) using Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit mAb #98134. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (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.
Western Blotting Image 1: Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit Monoclonal Antibody
Immunoprecipitation of cleaved caspase-8 protein from HeLa cell extracts. Cells were treated with Staurosporine #9953 (10 μM, 3 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit mAb. Western blot analysis was performed using Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Immunoprecipitation Image 1: Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit Monoclonal Antibody
Confocal immunofluorescent analysis of HCT 116 cells (left) or CRISPR/Cas9 Casp8 knockout (KO) HCT 116 cells (right), both serum starved and then treated with Staurosporine #9953 (1 μM, 4 hr) using Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit mAb (green), DyLight 650 Phalloidin #12956 (red), and DAPI #4083 (blue).
Immunofluorescence Image 1: Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 6

Cell Signaling Technologyロゴ
Flow cytometric analysis of serum starved HCT 116 cells, untreated (blue) or treated with Staurosporine #9953 (1 μM, 4 hr; green), using Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow Cytometry Image 1: Cleaved Caspase-8 (Asp374) (E6H8S) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.