Measured by its ability to reverse the inhibition of DEVD-AFC cleavage activity in cell extracts activated by addition of cytochrome c and dATP. The ED50 for this effect is 0.5-2.0 µM. Optimal dilutions should be determined by each laboratory for each application.
E. coli-derived human Livin protein Met1-Ser298, with a C-terminal 6-His tag
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
<1.5 EU per 1 μg of the protein by the LAL method.
34 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
38 kDa, reducing conditions
Read Publication using 1161-LV in the following applications:
EIA/RIA 96-well plate (Costar, Catalog # 3369) or equivalent
Fluorescence plate reader (Molecular Devices Model # SpectraMax Gemini EM) or equivalent
Preparation of Cell Extracts
Pellet cells from culture media by centrifugation at 1000 x g for 10 minutes at 4 °C.
Wash 2 times with PBS. Centrifudge as above and count cells before the final spin.
Add protease inhibitors to Extraction Buffer immediately prior to use. Final concentrations: 10 μg/mL Cytochalasin B, 2 ug/mL Chymostatin, 2 μg/mL Leupeptin, 2 μg/mL Antipain, 2 μg/mL Pepstatin, 100 μM PMSF and 1 mM DTT.
Solubilize the cells in ice cold Extraction Buffer at a density of 2 x 108 cells/mL.
Thoroughly resuspend the pellet by gently pipetting up and down. Incubate on ice for 10 minutes.
Pipette 200 μL aliquots into chilled microcentrifuge tubes.
Snap freeze in liquid nitrogen and store at ≤-70 °C. (Note: Freeze immediately at ≤ -70 °C if liquid nitrogen is unavailable to snap freeze.)
Activation of Caspase in Cell Extracts
Thaw cell extracts and centrifuge at 14,000 x g for 5 minutes at 4 °C. Transfer supernatants to chilled tubes and use within 1 hour.
Dilute rhLivin alpha (MW: 34 kDa) to various concentrations in Dilution Buffer. Make an initial dilution series of: 25,000, 12,500, 5000, 2500, 1250, 250, 125, and 25 nM. The final concentration range will be 10,000 to 10 nM in 25 μl total reaction volume.
Add 10 μL of cell extract to a tube containing 2.5 μL Cytochrome c, 2.5 μL of dATP and 10 μL Livin alpha dilution.
Total (no Livin alpha ) and inactive (no Livin alpha, Cytochrome-c, or dATP) controls should be run for each assay making up the volume difference with the appropriate buffer.
Incubate samples in a 30 °C water bath for 30 minutes.
To each well of a 96-well plate, add in the following order, 85 μL Assay Buffer and 5 μL of extracts activated in the presence or absence of added Livin alpha.
Start the reaction by adding 10 μL of 500 μM DEVD-AFC (50 μM final concentration).
Read at excitation and emission wavelengths 400 and 505 nm, respectively, in kinetic mode for 5 minutes.
Derive the 50% inhibiting concentration (IC50) of rhLivin alpha by plotting RFU/min vs. concentration with 4-PL fitting.
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Livin alpha Protein, CF
baculoviral IAP repeat containing 7
baculoviral IAP repeat-containing 7
KIAPRING finger protein 50
Kidney inhibitor of apoptosis protein
Melanoma inhibitor of apoptosis protein
MLIAPlivin inhibitor of apoptosis
RNF50LIVINbaculoviral IAP repeat-containing protein 7
Livin alpha is a member of the inhibitor of apoptosis (IAP) protein family and contains a single BIR and RING finger motif. The anti-apoptotic protein appears to inhibit the activation of Caspase-9 in cell extracts activated by cytochrome c and dATP. Livin alpha is the longer of two splice variants, encoding an additional 18 amino acid residues in the BIR-RING linking region.
Kasof, G.M. and B.C. Gomes (2001) J. Biol. Chem. 276(5):3238.
Vucic, D. et al. (2000) Curr. Biol. 10:1359.
Ashhab, Y. et al. (2001) FEBS 495:56.
Lin, J-H. et al. (2000) Biochem. Biophys. Res. Comm. 279:820.
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Survivin is thrivin' The survivin anti-apoptotic protein is the smallest member of a large family of proteins such as X-linked IAP, c-IAP1 and 2, IAP-like protein-2, melanoma IAP, Livin, and NAIP. Survivin regulates basic physiological events such as the cell cycle, tumor... Read full blog post.
Livin: On a Prayer Livin is a member of the inhibitor of apoptosis proteins (IAP) family that regulates programmed cell death. The Livin protein contains a single baculovirus IAP repeat (BIR) essential for function, along with a COOH-terminal RING-type zinc finger domai... Read full blog post.
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