Anti-human APAF-1 Monoclonal Antibody
Catalog #: 2340-MC-050
Size: 50 µg
APAF-1 (apoptotic protease activating factor-1) is a mammalian homolog of CED-4 that regulates cell death by participating in a ternary complex with cytochrome c, and procas-pase-9. APAF-1 is a member of the CARD protein family, which contains a caspase-recruitment domain linked to a nucleotide-binding domain (NBD), regulating apoptosis and/or NF-ê B activation. During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of APAF-1 and formation of the apoptosome caspase processing complex, triggering the postmitochondrial-mediated effector caspase cascade.
Immunogen: The antibody was raised against amino acids 10-214 of recombinant APAF-1.
Source: Mouse
Preparation: This antibody is affinity purified and provided in phosphate buffered saline without preservative.
Description: This monoclonal antibody, clone 94408.11, was produced from a murine hybridoma elicited from a mouse immunized with a recombinant fragment of human APAF-1 corresponding to amino acids 10-214 (accession number AF013263).
Physical State: Protein G affinity purified antibody at 1 mg/ml provided in phosphate buffered saline without preservative.
Specificity: This antibody detects human APAF-1.
Ig class: Mouse IgG2B
Storage: Store at -20°C. To avoid repeated freeze/thaws, freeze in working aliquots at -20°C.
Applications: Western blot.
For Western blot analysis, the recommended concentration is 1 µg/ml but empirical determination will be required for optimal results.
Immunoblots of SDS-extracts from 2 x 105 (lane 1) and 7 x 104 (lane 2) human Jurkat cells. Cells were solubilized in hot 2X SDS sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris (pH 6.8), 10% glycerol, 0.01 % bromophenol blue) at 2 x 106 - 1 x 107 cells/ml. The extracts were heated in a boiling water bath for 5 minutes followed by sonication with a probe sonicator with 3 - 4 second bursts of 5 - 10 seconds each. Cell extract was diluted in 1X SDS sample buffer and electrophoresed on a 5-15% gradient SDS-polyacrylamide gel and transferred to PVDF membrane. Membranes were incubated with 1 µg/ml anti-APAF-1 overnight at 4 °C, followed by detection using a peroxidase conjugated secondary antibody and chemiluminescence.
Procedure for Immunoblotting using Peroxidase Detection:
Transfer the electrophoresed proteins to nitrocellulose or PVDF membrane by Western transfer. Incubate the membrane for 1 hour at room temperature in 2% (w/v) nonfat dry milk in 25 mM Tris (pH 7.5), 0.15 M NaCl, 0.05% Tween 20.
Incubate the membrane overnight at 4°C in 1:1000 of antibody in 1% (w/v) nonfat dry milk in 25 mM Tris (pH 7.5), 0.15 M NaCl, 0.05% Tween 20. Empirical determination of primary antibody concentration will be required for optimal results.
Wash the membrane at room temperature for at least 15 minutes with 3 changes of 25 mM Tris (pH 7.5), 0.15 M NaCl, 0.05% Tween 20.
Incubate the membrane overngiht at 4 °C in 25 mM Tris (pH 7.5), 0.15 M NaCl, 0.05% Tween 20 containing a dilution of anti-mouse antibody conjugated to Horseradish peroxidase. Empirical determination of secondary antibody concentration will be required for optimal results.
Wash the membrane for at least 15 minutes with 3 changes of 25 mM Tris (pH 7.5), 0.15 M NaCl, 0.05% Tween 20, then rinse in water.
Develop peroxidase reaction using Trevigen’s Blue Membrane Solution (Cat# 4857-20- 13) or Peroxyglow chemiluminescence reagents (Cat#s 4855-20-13 and 4855-20-14).
Tween 20 is a registered trademark of ICI Americas, Inc., Wilmington, DE