WB
Western blot analysis of Caspase-8 on different lysates with Rabbit anti-Caspase-8 antibody at 1/2,000 dilution. Lane 1: Jurkat cell lysate, Lane 2: Jurkat treated with 25μM Etoposide for 5 hours cell lysate, Lane 3: Jurkat treated with 25μM Etoposide for 16 hours cell lysate, Lane 4: Jurkat treated with 1μM staurosporine for 3 hours cell lysate, Lane 5: HeLa cell lysate, Lane 6: HeLa treated with 1μM staurosporine for 3 hours cell lysate, Lane 7: HeLa treated with 100μM Etoposide for 4 hours cell lysate, Lysates/proteins at 30 µg/Lane. Exposure time: 3 minutes; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/2,000 dilution was used in antibody diluent at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.ICC/IF
Immunocytochemistry analysis of HeLa cells treated with or without 1μM staurosporine for 3 hours labeling Caspase-8 with Rabbit anti-Caspase-8 antibody at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Caspase-8 antibody at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594) was used as the secondary antibody at 1/1,000 dilution.IHC
Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-Caspase-8 antibody at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.FC
Flow cytometric analysis of HeLa cells treated with or without 1μM staurosporine for 3 hours cells labeling Caspase-8. Cells were fixed and permeabilized. Then stained with the primary antibody (1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).IP
Caspase-8 was immunoprecipitated from 0.2 mg HeLa treated with 1μM staurosporine for 3 hours cell lysate with Rabbit anti-Caspase-8 antibody at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using Rabbit anti-Caspase-8 antibody at 1/2,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa treated with 1μM staurosporine for 3 hours cell lysate (input), Lane 2: Rabbit anti-Caspase-8 antibody IP in HeLa treated with 1μM staurosporine for 3 hours cell lysate, Lane 3: Rabbit IgG instead of Rabbit anti-Caspase-8 antibody in HeLa treated with 1μM staurosporine for 3 hours cell lysate, Blocking/Dilution buffer: 5% NFDM/TBST. Exposure time: 1 minute 28 seconds.| Product Name | Caspase-8 Recombinant Rabbit Monoclonal Antibody |
|---|---|
| Antibody Type | Primary Antibodies |
| Product description | Initiator caspases, which include caspase-8, activate effector caspases by cleaving inactive forms of effector caspases. In the activation cascade responsible for apoptosis induced by TNFRSF1A and mediated by TNFRSF6/FAS, caspase-8 is the most upstream protease. Caspase-8 binds to adaptor molecule FADD, forming an aggregate referred to as death-inducing signaling complex (DISC), which activates caspase-8. The actived protein is released from the complex and further activates downstream apoptotic proteases. Caspase-8, which is a heterodimer consisting of two subunits (p18 and p10), is widely expressed, but is detected at highest levels in peripheral blood leukocytes (PBLs), thymus, liver and spleen. Defects in CASP8, the gene encoding for caspase-8, may cause CASP8D (caspase-8 deficiency disorder), which is characterized by splenomegaly and CD95-induced apoptosis of PBLs, may lead to immunodeficiency due to defects in T lymphocyte, NK cell and B lymphocyte activation. |
| Immunogen | Synthetic peptide within human Caspase-8 aa 325-374 / 479. |
| Clonality | Polyclonal |
|---|---|
| Isotype | IgG |
| Host Species | Recombinant rabbit |
| Tested Applications | FCICC/IFIHCIPWB |
| WB:1:1000-1:2000 ICC/IF:1:100 IHC:1:200-1:1000 FC:1:1000 IP:1-2μg/sample |
|
| Species Reactivity | Human |
| Concentration | 1mg/ml |
| Purification | Protein A |
| Alternative Names | ALPS2B antibody Amyotrophic lateral sclerosis 2 chromosomal region candidate gene 12 protein antibody Apoptotic cysteine protease antibody Apoptotic protease Mch-5 antibody Apoptotic protease Mch5 antibody CAP4 antibody CASP-8 antibody CASP8 antibody CASP8_HUMAN antibody Caspase 8 antibody Caspase 8 apoptosis related cysteine peptidase antibody Caspase-8 subunit p10 antibody CED 3 antibody FADD Like ICE antibody FADD-homologous ICE/CED-3-like protease antibody FADD-like ICE antibody FLICE antibody FLJ17672 antibody ICE-like apoptotic protease 5 antibody MACH alpha 1/2/3 protein antibody MACH antibody MACH beta 1/2/3/4 protein antibody MCH5 antibody MGC78473 antibody MORT1 associated ced 3 homolog antibody MORT1-associated CED-3 homolog antibody OTTHUMP00000163717 antibody OTTHUMP00000163720 antibody OTTHUMP00000163724 antibody OTTHUMP00000163725 antibody OTTHUMP00000165062 antibody OTTHUMP00000165063 antibody OTTHUMP00000165064 antibody OTTHUMP00000206552 antibody OTTHUMP00000206582 antibody |
|---|---|
| Molecular Weight(MW) | 55kDa(Observed band size: 18-55kDa) |
| Cellular Localization | Cytoplasm, Nucleus. |
| SwissProt ID | Q14790 |
|---|
WB
Western blot analysis of Caspase-8 on different lysates with Rabbit anti-Caspase-8 antibody at 1/2,000 dilution. Lane 1: Jurkat cell lysate, Lane 2: Jurkat treated with 25μM Etoposide for 5 hours cell lysate, Lane 3: Jurkat treated with 25μM Etoposide for 16 hours cell lysate, Lane 4: Jurkat treated with 1μM staurosporine for 3 hours cell lysate, Lane 5: HeLa cell lysate, Lane 6: HeLa treated with 1μM staurosporine for 3 hours cell lysate, Lane 7: HeLa treated with 100μM Etoposide for 4 hours cell lysate, Lysates/proteins at 30 µg/Lane. Exposure time: 3 minutes; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/2,000 dilution was used in antibody diluent at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of HeLa cells treated with or without 1μM staurosporine for 3 hours labeling Caspase-8 with Rabbit anti-Caspase-8 antibody at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Caspase-8 antibody at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594) was used as the secondary antibody at 1/1,000 dilution.
IHC
Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-Caspase-8 antibody at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
FC
Flow cytometric analysis of HeLa cells treated with or without 1μM staurosporine for 3 hours cells labeling Caspase-8. Cells were fixed and permeabilized. Then stained with the primary antibody (1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
IP
Caspase-8 was immunoprecipitated from 0.2 mg HeLa treated with 1μM staurosporine for 3 hours cell lysate with Rabbit anti-Caspase-8 antibody at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using Rabbit anti-Caspase-8 antibody at 1/2,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa treated with 1μM staurosporine for 3 hours cell lysate (input), Lane 2: Rabbit anti-Caspase-8 antibody IP in HeLa treated with 1μM staurosporine for 3 hours cell lysate, Lane 3: Rabbit IgG instead of Rabbit anti-Caspase-8 antibody in HeLa treated with 1μM staurosporine for 3 hours cell lysate, Blocking/Dilution buffer: 5% NFDM/TBST. Exposure time: 1 minute 28 seconds.| Application Notes | WB:1:1000-1:2000 ICC/IF:1:100 IHC:1:200-1:1000 FC:1:1000 IP:1-2μg/sample |
|---|
| Form | Liquid |
|---|---|
| Storage Instructions | Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles. |
| Storage Buffer | 1*TBS (pH7.4), 1%BSA, 40%Glycerol. Preservative: 0.05% Sodium Azide. |
