cleaved Caspase-9 Rabbit Recombinant mAb

Catalog No.A5074

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  1. 1. Why choose rabbit monoclonal antibody?
  2. Rabbit monoclonal antibody has over 100 times higher affinity than that of mouse monoclonal.
  3. 2. Why choose recombinant antibodies?
  4. Recombinant antibodies are known for higher purity and minimal deviation between batches, versus regular antibodies.

Usage Information

Application WB,ELISA
Dilution
WB
1:1000
Reactivity Human Mouse
MW (kDa) 46, 39, 37, 35kDa
Source Rabbit
Concentration 1mg/ml
Storage buffer 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.
Storage Store at –20°C.

Protocol

WB
+ Expand

Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

Description

Specificity cleaved Caspase-9 Rabbit Recombinant mAb detects endogenous level of cleaved Caspase-9.
Background Caspases are a family of endoproteases that provide critical links in cell regulatory networks controlling inflammation and cell death. Caspases have been broadly classified by their known roles in apoptosis (caspase-3, -6, -7, -8, and -9 in mammals), and in inflammation (caspase-1, -4, -5, -12 in humans and caspase-1, -11, and -12 in mice). Caspases involved in apoptosis have been subclassified by their mechanism of action and are either initiator caspases (caspase-8 and -9) or executioner caspases (caspase-3, -6, and -7). Caspases are initially produced as inactive monomeric procaspases that require dimerization and often cleavage for activation. The initiator caspase responsible for the intrinsic apoptosis pathway is caspase-9, which is activated by dimerization induced when the caspase-9 CARD domain binds to the adapter protein apoptotic protease-activating factor-1 (APAF1). Caspase-9 is activated by association with the Apaf-1 (apoptotic protease-activating factor-1) apoptosome complex, cleaves and activates the downstream effector caspases-3 and -7, thereby executing the caspase-cascade and cell-death programme. Caspase-9 cleavage allows the initiation of the caspase cascade to be very tightly controlled, and both cleavage events are important.

Datasheet & MSDS

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Caspase Signaling Pathway Map

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