Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF

Catalog #: 8646-DH Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
8646-DH-050

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Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to produce NADH during the oxidation of lipoic acid. The specific activity is >5,000 pmol/min/μg, as measured under the described conditions.

Source

E. coli-derived human Dihydrolipoamide Dehydrogenase/DLD protein

GGS	HHHHHH	GMASLENLYFQ	Human DLD (Ala36-Phe509) Accession # P09622
N-terminus			C-terminus

Ala36 - Phe509, with N-terminal GGS-6-His tag and GMASLENLYFQ insertion

Accession #

P09622

N-terminal Sequence
Analysis

Gly

Predicted Molecular Mass

52 kDa

SDS-PAGE

52-62 kDa, reducing conditions

Product Datasheets

8646-DH

Product Datasheet

COA

SDS

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.

8646-DH

Formulation	Supplied as a 0.2 μm filtered solution in Sodium Phosphate and Sucrose.
Shipping	The product is shipped with polar packs. 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

Materials

Assay Buffer: 50 mM Sodium Phosphate, 1 mM EDTA, 1 mg/mL BSA, pH 5.5
Recombinant Human Dihydrolipoamide Dehydrogenase/DLD (rhDLD) (Catalog # 8646-DH)
NADH (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
NAD (Sigma, Catalog # N6522), 100 mM stock in deionized water
(±)-alpha -Lipoic acid (Sigma, Catalog # T1395), 20 mM stock in 95% Ethanol
96-well Clear Plate (Catalog # DY990)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Create a Substrate Mixture containing 0.4 mM NADH, 0.2 mM NAD and 2 mM Lipoic Acid in Assay Buffer.
Incubate Substrate Mixture at room temperature for 5 minutes in the dark.
Dilute rhDLD to 2 µg/mL in Assay Buffer.
Load 50 µL of 2 µg/mL rhDLD in a plate, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
Read plate in kinetic mode for 5 minutes at an absorbance of 340 nm.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (OD/min) x -1 x well volume (L) x 10¹² pmol/mol
	ext. coeff (M^-1cm^-1) x path corr.* (cm) x amount of enzyme (µg)

*Adjusted for Substrate Blank
** Using the extinction coefficient 6220 M^-1cm^-1
*** Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD

Per Well:

rhDLD: 0.1 µg
NADH: 0.2 mM
NAD: 0.1 mM
Lipoic Acid: 1 mM

Reconstitution Calculator

Background: Dihydrolipoamide Dehydrogenase/DLD

Dihydrolipoamide dehydrogenase (DLD), also known as LADH, is an NAD-dependent oxidoreductase in the mitochondrial matrix (1). DLD serves as the E3 subunit of four mitochondrial enzyme complexes: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, branched chain alpha-ketotacid dehydrogenase, and the glycine cleavage system (2, 3). It is active as a 120 kDa dimer that catalyzes oxidation within these enzyme complexes. Several mutations of human DLD have been described, some of which contribute to the loss of respiratory function during oxidative stress (4, 5). DLD mutations located at the interface between dimer subunits can impair dimer formation (5, 6). The involvement of DLD in the regulation of lipid peroxidation and lactate metabolism is important for mouse hippocampal neuroblast proliferation and hamster sperm capacitation, respectively (7, 8). DLD polymorphisms in insects can increase their resistance to the pesticide phosphine gas, while they can increase the sensitivity to arsenite in the nematode C. elegans (9). Mature human DLD shares 95-96% amino acid (aa) sequence identity with hamster, mouse, and rat DLD. Alternative splicing generates additional human DLD isoforms that lack the N-terminal 99 aa or carry a deletion of aa 147-194.

References

Brand, M.D. (2010) Exp. Gerontol. 45:466.
Johnson, M.T. et al. (1997) Proc. Natl. Acad. Sci. USA 94:14512.
Sundquist, A.R. and R.C. Fahey (1988) J. Bacteriol. 170:3459.
Ambrus, A. et al. (2011) Hum. Mol. Genet. 20:2984.
Vaubel, R.A. et al. (2011) J. Biol. Chem. 286:40232.
Babady, N.E. et al. (2007) Proc. Natl. Acad. Sci. USA 104:6158.
Calingasan, N.Y. et al. (2008) Neuroscience 153:986.
Panneerdoss, S. et al. (2012) J. Androl. 33:699.
Schlipalius, D.I. et al. (2012) Science 338:807.

Entrez Gene IDs

1738 (Human); 13382 (Mouse); 298942 (Rat)

Alternate Names

2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex); branched chain keto acid dehydrogenase complex; Diaphorase; dihydrolipoamide dehydrogenase (E3 component of pyruvate dehydrogenase complex; Dihydrolipoamide Dehydrogenase; dihydrolipoamide dehydrogenasemitochondrial; DLD; DLDD; DLDH; E3 component of pyruvate dehydrogenase complex, 2-oxo-glutarate complex; E3; EC 1.8.1; EC 1.8.1.4; GCSL; Glycine cleavage system L protein; glycine cleavage system protein L; LAD; Lipoamide Dehydrogenase; lipoamide reductase; lipoyl dehydrogenase; PHE3