Recombinant Human Livin alpha Protein, CF

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R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human Livin alpha Protein, CF Summary

Product Specifications

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.5 EU per 1 μg of the protein by the LAL method.
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
Accession #
N-terminal Sequence
Structure / Form
Predicted Molecular Mass
34 kDa
38 kDa, reducing conditions

Product Datasheets

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Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.


Formulation Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT and Glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure


Preparation of Cell Extracts

  • Jurkat A3 wild type cells (ATCC # CRL-2570)
  • Phosphate Buffered Saline (PBS, pH 7.4)
  • Extraction Buffer: 50 mM HEPES-KOH (pH 7.5), 10 mM KCl, 5 mM EGTA, 1 mM MgCl2, 0.2% CHAPS, 0.2 mM DTT
  • Protease inhibitors: Cytochalasin B (Sigma # C6762), Chymostatin (Sigma # C7268), Leupeptin (Sigma # L8511), Antipain (Sigma # A6191), Pepstatin (Sigma # P4265), PMSF (Sigma # P7626)

Activation of Caspase in Cell Extracts

  • Cytochrome c, Bovine heart: (Sigma, Catalog # C3131) 2 mg/mL stock in deionized water
  • dATP (Sigma, Catalog # D6500), 10 mM stock adjusted to pH 7.5 with KOH
  • Recombinant Human Livin alpha (rhLivin alpha ) (Catalog # 1161-LV)
  • Dilution Buffer: 25 mM HEPES (pH 8.0), 0.1 M NaCl, 1 mM DTT, 10% Glycerol
  • Assay Buffer: 10 mM HEPES (pH 7.5), 0.5 mM EGTA, 5 mM DTT, 10% Glycerol
  • Substrate: Ac-Asp-Glu-Val-Asp-AFC (DEVD-AFC, MP Biomedicals, Catalog # AFC138) 500 μM stock in DMSO
  • 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

  1. Pellet cells from culture media by centrifugation at 1000 x g for 10 minutes at 4 °C.
  2. Wash 2 times with PBS. Centrifudge as above and count cells before the final spin.
  3. 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.
  4. Solubilize the cells in ice cold Extraction Buffer at a density of 2 x 108 cells/mL.
  5. Thoroughly resuspend the pellet by gently pipetting up and down. Incubate on ice for 10 minutes.
  6. Pipette 200 μL aliquots into chilled microcentrifuge tubes.
  7. 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

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. Incubate samples in a 30 °C water bath for 30 minutes.
  6. 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.
  7. Start the reaction by adding 10 μL of 500 μM DEVD-AFC (50 μM final concentration).
  8. Read at excitation and emission wavelengths 400 and 505 nm, respectively, in kinetic mode for 5 minutes.
  9. Derive the 50% inhibiting concentration (IC50) of rhLivin alpha by plotting RFU/min vs. concentration with 4-PL fitting.

Reconstitution Calculator

Reconstitution Calculator

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Background: Livin

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.

  1. Kasof, G.M. and B.C. Gomes (2001) J. Biol. Chem. 276(5):3238.
  2. Vucic, D. et al. (2000) Curr. Biol. 10:1359.
  3. Ashhab, Y. et al. (2001) FEBS 495:56.
  4. Lin, J-H. et al. (2000) Biochem. Biophys. Res. Comm. 279:820.
Entrez Gene IDs
79444 (Human)
Alternate Names
baculoviral IAP repeat containing 7; baculoviral IAP repeat-containing 7; BIRC7; KIAP; KIAPRING finger protein 50; Kidney inhibitor of apoptosis protein; livin inhibitor-of-apoptosis; Livin; Melanoma inhibitor of apoptosis protein; ML-IAP; MLIAPlivin inhibitor of apoptosis; ML-IAPmliap; RNF50LIVINbaculoviral IAP repeat-containing protein 7

Citation for Recombinant Human Livin alpha Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1

  1. Soluble PD-L1 as a biomarker in malignant melanoma and checkpoint blockade
    Authors: J Zhou, KM Mahoney, A Giobbie-Hu, F Zhao, S Lee, X Liao, S Rodig, J Li, X Wu, LH Butterfiel, M Piesche, MP Manos, LM Eastman, G Dranoff, GJ Freeman, FS Hodi
    Cancer Immunol Res, 2017;0(0):.
    Species: Human
    Sample Types: Antibody
    Applications: ELISA (Standard)


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