Caspase-3 Fluorometric Assay Kit

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General description

Product name

Caspase-3 Assay Kit (Fluorometric)

Detection method: Fluorescent

Example type

Tissue extracts, cell lysate

Test type: Enzymatic activity

Test time: 2h 00m

Product description

The Caspase-3 (Fluorometric) Assay Kit (ab39383) provides a simple and convenient means of testing DVD-dependent caspase activity. The Caspase-3 assay protocol is based on the detection of cleavage of the substrate DVD-AFC (AFC: 7-amino-4-trifluoromethylcoumarin). DVD-AFC emits blue light (λ max = 400 nm).

On cleavage of the substrate by CPP32 or related caspases, free CFA emits a greenish-yellow fluorescence (Ex / Em = 400/505 nm). The signal can be quantified using a fluorometer or fluorescence microtiter plate reader. Comparison of the CFA fluorescence of an apoptotic sample with an uninduced control allows the determination of the fold increase in Caspase-3 / CPP32 activity.

Caspase-3 assay protocol summary:

  • lyse cells / homogenize and lyse tissues in lysis buffer
  • incubate on ice for 10 min
  • add reaction buffer and DVD-AFC substrate and incubate for 1-2 hours at 37ºC
  • analyze with fluorometer or microplate reader

Notes

Due to the nature of the substrate, this assay also detects caspase-7 activity.

Other caspase and apoptosis assays

Abcam has not applied for and does not intend to apply for REACH Authorization for the use of products by customers containing substances on the European Authorization List (Annex XIV). It is the responsibility of our clients to verify the need to apply the REACH Authorization, and any other relevant authorization, for the intended uses.

Platform: Microplate reader

Properties

Storage instructions

Store at -20 ° C. See protocols.

Function

Involved in the caspase activation cascade responsible for the execution of apoptosis. At the onset of apoptosis, it proteolytically cleaves poly (ADP-ribose) polymerase (PARP) at a ‘216-Asp-Gly-217 ‘link. It cleaves and activates the sterol regulatory element-binding proteins (SREBPs) between the basic helix-loop-helix leucine zipper domain and the membrane-binding domain. Cleavage and activate caspase-6, -7, and -9. Involved in huntingtin cleavage.

Tissue specificity

Highly expressed in the lung, spleen, heart, liver, and kidney. Moderate levels in the brain and skeletal muscle and low in testes. It is also found in many cell lines, the highest expression in the cells of the immune system.

Sequence similarities

It belongs to the C14A peptidase family.

Post-translational modifications

Cleavage by granzyme B, caspase-6, caspase-8, and caspase-10 generates the two active subunits. Additional processing of the propeptides is likely due to the autocatalytic activity of the activated protease. Active heterodimers also appear between the small subunit of caspase-7 protease and the large subunit of caspase-3 and vice versa. S-nitrosylated at its catalytic site cysteine ​​in unstimulated human cell lines and denitrosylated after activation of the apoptotic Fas pathway, associated with an increase in intracellular caspase activity. Thus, Fas activates caspase-3 not only by inducing caspase zymogen cleavage into its active subunits but also by stimulating denitrosylation of its thiol active site.

Cell location:

  • Cytoplasm.
  • Previous Destination Information for UniProt Access
  • The UniProt Consortium

Alternative names

  • A830040C14Rik
  • Apopain
  • CASP 3

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