Download BD Pharmingen™ Polyclonal Rabbit Anti-Human

BD Pharmingen™
Bioimaging Certified Reagent
Technical Data Sheet
Polyclonal Rabbit Anti-Human Caspase-8
Product Information
Material Number:
551234
Alternate Name:
FLICE, MACH-1, Mch5
Reactivity:
QC Testing: Human
Component:
Description:
Size:
Immunogen:
Target MW:
Storage Buffer:
51-9000095
Polyclonal Rabbit Anti-Human Caspase-8 (FLICE, MACH-1, Mch5)
Component:
Description:
Size:
Concentration:
Storage Buffer:
51-16526N
Jurkat Cell Lysate
100 µl (1 ea)
Human Caspase-8 aa 2-20 Peptide
50/55 kD, 36/40 kD, and 23 kD
Aqueous buffered solution containing ≤0.09% sodium azide.
50 µg (1 ea)
1.0 mg/ml
1 mg/ml in SDS-PAGE buffer (62mM Tris pH 6.8, 2% SDS, 0.9%
b-mercaptoethanol, 0.003% bromophenol blue, 5% glycerol)
Description
Caspase-8 is a cytosolic protein with homology to the CD95/Fas-associated signal transducer, FADD/MORT-1, as well as to other caspase
(ICE/Ced-3) cysteine proteases. Caspase-8 is produced as a proenzyme which upon receptor aggregation is proteolytically cleaved into
smaller subunits of 40/36 kDa (doublet) and 23 kDa subunits. These subunits form a proteolytically active heterodimer capable of cleaving
other caspase family members, as well as substrates such as PARP. This polyclonal antibody recognizes both the proform of caspase-8 as a
55/50 kDa doublet as well as the cleaved forms which migrate at 40/36 kDa (doublet) and 23 kDa in SDS/PAGE.
This antibody is routinely tested by Western blot analysis and immunofluorescent imaging. Other applications were tested at BD Biosciences
Pharmingen during antibody development only or reported in the literature.
Preparation and Storage
The polyclonal antibody was purified from antiserum by negative adsorption and affinity chromatography.
Store the polyclonal antibody undiluted at 4°C.
Store the Jurkat cell lysate undiluted at -20°C.
Application Notes
Application
Bioimaging
Routinely Tested
Western blot
Routinely Tested
Recommended Assay Procedure:
Methanol Procedure for a 96 well plate:
Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and
add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking
buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at
RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image
sample.
Triton-X 100 Procedure for a 96 well plate:
Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and
add 100 µl/well 0.1% Triton-X 100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well
blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1
hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three
times with PBS. Image sample.
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Western blot analysis of caspase-8. Lysates from
control (lane 1) or apoptotic (lane 2) Jurkat cells were
probed with the anti-caspase-8 antibody at a dilution of
1:3000. The proform of caspase-8 migrates at ~55/50 kDa
(doublet), while the cleaved forms migrate at ~40/36
(doublet) and ~23 kDa.
Immunofluorescent staining of U2OS cells. Cells were
seeded in a 96 well imaging plate (Cat. No. 353219) at ~
10,000 cells per well. After overnight incubation, cells
were stained using the Triton X100 fix/perm protocol (see
Recommended Assay Procedure) and the anti- caspase-8
antibody. The second step reagent was FITC goat antirabbit Ig (Cat. No. 554020). Images were taken on a
Pathway 850 imager using a 20x objective. This antibody
also stained HeLa and A549 cells and worked with both
the Triton X100 and Methanol fix/perm protocols (see
Recommended Assay Procedure).
Suggested Companion Products
Catalog Number
Name
Size
Clone
554021
HRP Goat Anti-Rabbit Ig
1.0 ml
(none)
554020
FITC Goat Anti-Rabbit Ig
0.5 mg
(none)
550959
Jurkat Apoptotic Lysate Set I
500 µg
(none)
611451
Jurkat Cell Lysate
500 µg
(none)
Product Notices
1.
Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2.
Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
3.
Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before
discarding to avoid accumulation of potentially explosive deposits in plumbing.
References
Boesen-de Cock JG, Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging
stimuli etoposide and gamma-radiation downstream from caspase-8 activation. J Biol Chem. 1999; 274(20):14255-14261.(Biology: Western blot)
Cock JG, Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. CD95 (Fas/APO-1) induces ceramide formation and apoptosis in the absence of a functional acid
sphingomyelinase. J Biol Chem. 1998; 273(13):7560-7565.(Immunogen: Western blot)
Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing
signaling complex. Cell. 1996; 85(6):817-827.(Biology)
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