Recombinant Human ALDH1A2 His-tag, CF

Catalog # Availability Size / Price Qty
10132-DH-020
ALDH1A2 Protein Enzyme Activity
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Recombinant Human ALDH1A2 His-tag, CF Summary

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by the ability to catalyze the oxidation of 4-nitrobenzaldehyde. The specific activity is >150 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human ALDH1A2 protein
Thr2-Ser518
with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Analysis
Met1
Predicted Molecular Mass
58 kDa
SDS-PAGE
58 kDa, under reducing conditions

Product Datasheets

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.

10132-DH

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT.
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

Materials
  • Assay Buffer: 50 mM Tris, 100 mM KCl, 2 mM DTT, pH 8.5
  • Recombinant Human ALDH1A2 (rhALDH1A2) (Catalog # 10132-DH)
  • beta -Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
  • 4-Nitrobenzaldehyde (4-NBA) (Sigma, Catalog # 72800), 200 mM stock in DMSO
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhALDH1A2 to 10 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 2 mM beta -NAD and 2 mM 4-NBA in Assay Buffer.
  3. Load 50 µL of 10 µg/mL rhALDH1A2 into the plate, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate Mixture.
  4. Read plate at 340 nm (absorbance) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted Vmax* (OD/min) 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 0.320 cm
Note: the output of many spectrophotometers is in mOD

Per Well:
  • rhALDH1A2: 0.5 µg
  • beta -NAD: 1 mM
  • 4-NBA: 1 mM

Data Image

SDS-PAGE <br> View Larger

1 μg/lane of Recombinant Human ALDH1A2 His-tag was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by silver staining, showing a band at 58 kDa under reducing conditions.

Reconstitution Calculator

Reconstitution Calculator

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Background: ALDH1A2

Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent enzymes that detoxify aldehydes by oxidizing them to carboxylic acids. Nineteen ALDHs are present in humans, expressed in a variety of organelles and having different substrate preferences (1). Human ALDH1A2 is a NAD-dependent, cytosolic member of the ALDH1A subfamily class 1 that forms active tetramers that synthesize retinoic acid from retinal.  Each monomer has three functional regions: a catalytic domain containing the active site cysteine, an NAD-binding domain, and the oligomerization domain (2, 3). ALDH1A2 exhibits the highest substrate specificity and catalytic efficiency for retinal oxidation to retinoic acid (RA) (4) and thus plays an important role in regulation of RA production and signaling. ALDH1A2 null mice are embryonic lethal (5) and mutations in ALDH1A2 are associated with pathological conditions such as osteoarthris (6-8). Additionally, ALDH1A2 is suggested to play a role in several cancers. ALDH1A2 was a suggested tumor marker (9, 10) and predictor of prostate cancer relapse (11). Abnormally low levels of ALDH1A2 has been observed in several cancers including breast (12), squamous cell carcinoma of the head and neck (13), and specifically in high grade ovarian cancer, suggesting high expression may be associated with favorable prognosis (14).  Overexpression of ALDH1A2 in cancer lines resulted in decreased proliferation and migration (14) supporting that ALDH1A2 plays a tumor suppressor role. In contrast, ALDH1A2 is reported to exhibit high expression correlated with worse overall survival in non-small-cell lung cancer (15) and chemoresistant cancer stem cells in neuroblastoma (16, 17). Pharmacologically specific inhibitors and activators of ALDH1A2 are of interest due to its implied role in a variety of diseases and proliferation and drug resistance (1, 3, 15, 18).

References
  1. Koppaka, V. et al. (2012) Pharmacol. Rev. 64:520.
  2. Perez-Miller, S. J. and T. D. Hurley (2003) Biochemistry. 42:7100.
  3. Chen, Y. et al. (2018) ACS Chem. Biol. 13:582.
  4. Duester, G. (2001) Chem. Biol. Interact. 130:469.
  5. Niedereither, K. et al. (1999) Nat. Genet. 21:444.
  6. Pavan, M. et al. (2009) B.M.C. Med. Genet. 10:113.
  7. Styrkarsdottir, U. et al. (2014) Nat. Genet. 46:498.
  8. Shepherd, C. et. al. (2018) Arthritis Rheumatol. 70:1577.
  9. Kim, H. et al. (2005) Cancer Res. 65:8118.
  10. Touma, S. E. et al. (2009) Biochem. Pharmacol. 78:1127.
  11. Nim, H. T. et al. (2017) Front. Oncol. 7:30.
  12. Mira, Y. L. R. et al. (2000) J. Cell Physiol. 185:302.
  13. Seidensaal, K. et al. (2015) Mol. Cancer 14:204.
  14. Wang, Y. et al. (2018) Onco. Targets Ther. 31:599.
  15. You, Q. et al. (2015) Drug Des. Devel. Ther. 9:5087.
  16. Hartomo, T. B. et al. (2015) Int. J. Oncol. 46:1089.
  17. Singh, S. et al. (2015) Adv. Exp. Med. Biol. 815:281.
  18. Moreb, J. S. et al. (2012) Chem. Biol. Interact. 195:52.
Long Name
Aldehyde Dehydrogenase 1 Family, Member A2
Entrez Gene IDs
8854 (Human); 19378 (Mouse)
Alternate Names
aldehyde dehydrogenase 1 family, member A2; Aldehyde dehydrogenase family 1 member A2; ALDH 1A2; ALDH1A2; EC 1.2.1; EC 1.2.1.36; RALDH 2; RALDH; RALDH(II); RALDH2; RALDH2MGC26444; RALDH2-T; retinal dehydrogenase 2; Retinaldehyde-specific dehydrogenase type 2

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