Caspase-3 Rabbit Recombinant mAb

Catalog No.A5013

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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.

WB

Validated by Selleck

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Usage Information

Application WB,ELISA
Dilution
WB
1:1000
Reactivity Human Rat
MW (kDa) 32kDa
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 Caspase-3 Rabbit Recombinant mAb detects endogenous levels of total Caspase-3
Background Caspase-3 is a member of cysteine-aspartic acid protease (caspase) family that are involved in the signaling pathways of apoptosis, necrosis and inflammation. Caspases exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. This protein cleaves and activates caspases 6, 7 and 9, and the protein itself is processed by caspases 8, 9 and 10. It is the predominant caspase involved in the cleavage of amyloid-beta 4A precursor protein, which is associated with neuronal death in Alzheimer's disease.

Datasheet & MSDS

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Caspase Signaling Pathway Map

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