Calpain Activity Assay Kit

Antigen: Calpain

Synonyms: CG18152, EST C, DmelCG7563, CG7563, CAPN1, calpain, Tb08.28A12.350

Reactivity: Please enquire

Application: Biochemical Assay (BCA)

Catalog no.: ABIN411643

Additional Information

Sample Type: Cell and Tissue Culture Lysate

Components: Extraction Buffer, 10X Reaction Buffer, Calpain Substrate Ac-LLY-AFC , Active Calpain I (Positive Control), Calpain Inhibitor Z-LLY-FMK

Description: Activation of calpain is involved in many forms of physiological and pathological processes (e.g., apoptosis). Calpain activation requires cell membrane and Ca 2+ , and activated calpain is released into cytosol. The Calpain Activity Assay Kit provides optimized buffers and reagents for a convenient measurement of calpain activity. The Extraction Buffer provided with the kit specifically extracts cytosolic proteins without contaminations of cell membrane and lysosome proteases. The Extraction Buffer also prevents auto-activation of calpain during the extraction procedure. Thus, the kit detects only activated calpain in cytosol upon treatment of cells with inducers (e.g., chemicals or drugs). The fluorometric assay is based on the detection of cleavage of calpain substrate Ac-LLY-AFC. Ac-LLY-AFC emits blue light ( λ max = 400 nm), upon cleavage of the substrate by calpain, free AFC emits a yellow-green fluorescence ( λ max = 505 nm), which can be quantified using a fluorometer or a fluorecence plate reader. Comparison of the fluorescence intensity from a treated sample with a normal control allows determination of the changes in calpain activity.

Application Details

Protocol: 1. Treat cells by desired methods. Concurrently incubate a control culture without treatment. 2. Count cells and pellet ~1-2 x 10 6 cells by centrifugation. 3. Resuspend cells in 100 μ l Extraction Buffer and incubate samples on ice for 20 minutes. Gently mix the samples by tapping several times during incubation. 4. Centrifuge for 1 min in a microcentrifuge (10,000 g) and transfer supernatant to a fresh tube and put on ice. Assay protein concentration (

Application Notes: Note: because of the high reducing agent content in the extraction buffer-dilute about 10-fold then use a Coomassie-based protein assay -such as BioVision’s Cat#810-1000). 5. Dilute the cell lysate (~50-200 μ g) to 85 μ l of Extraction Buffer. For positive control, add 1-2 μ l Active Calpain to 85 μ l of Extraction Buffer. For negative control, use untreated cell lysate or add 1 μ l Calpain Inhibitor to the treated cell lysate. 6. Add 10 μ l of 10X Reaction Buffer and 5 μ l of Calpain Substrate to each assay. 7. Incubate at 37 o C for 1 hour in the dark. 8. Read samples in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. For a plate reading set-up, transfer the samples to a 96-well plate. 9. The changes in calpain activity can be determined by comparing results of treated samples and negative control. Alternatively, the activity can be expressed as Relative Fluorescent Unit (RFU) per milligram protein of each sample.

Storage: -70°C

Storage Comment: Store kit at –70 °C (Store Extraction Buffer and 10X Reaction Buffer at 4 °C after opening).

Expiry Date: 6-12 months

Restrictions: For Research Use only

Publications

Huang, Zhou, Saberwal et al.: "Interferon consensus sequence binding protein (ICSBP) decreases beta-catenin activity in myeloid cells by repressing GAS2 transcription." in: Molecular and cellular biology, Vol. 30, Issue 19, pp. 4575-94, 2010 (PubMed).

Yeh, Kuo, Chan et al.: "Transforming growth factor-{beta} and oxidative stress mediate tachycardia-induced cellular remodeling in cultured atrial-derived myocytes." in: Cardiovascular research, 2011 (PubMed).

Product: Li, Okumura, Nomura et al.: "Oxidatively damaged proteins in the early stage of testicular toxicities in male rats by orally administered with a synthetic oestrogen, diethylstilbestrol." in: Reproductive toxicology (Elmsford, N.Y.), Vol. 31, Issue 1, pp. 26-34, 2011 (PubMed).

Shinde, Sizova, Lin et al.: "ER Stress in Retinal Degeneration in S334ter Rho Rats." in: PLoS ONE, Vol. 7, Issue 3, pp. e33266, 2012 (PubMed).