Aldolase C Protein, Human (HEK293, His)

Aldolase C Protein, Human (HEK293, His) is a recombinant human Fructose-bisphosphate aldolase C produced in HEK293 cells, with a His tag at the C-terminus. Aldolase C (ALDOC), which belongs to the aldolase family, is found in normal brain tissue and is responsible for the repair of injured tissue^[1].

The aldolases family is one of the main enzymes involved in the process of glycolysis. Aldolase C (ALDOC) is found in normal brain tissue and is responsible for the repair of injured tissue ^[1].

The specific activity is >4 U/mg. One unit will convert 1.0 µmol of fructose 1,6-diphosphate to dihydroxyacetone phosphate and glyceraldehydes 3- phosphate per minute at pH 7.5 at 37 °C.

Materials
Aldolase C Protein, Human (HEK293, His) (HY-P7478): Dissolve in deionized water to a concentration of 10 μg/mL.
Substrate: Fructose 1,6-bisphosphate. Accurately weigh 16.24 mg and dissolve in 1 mL of deionized water to a concentration of 40 mM.
NADH: Accurately weigh 5.672 mg of solid powder and dissolve in 1 mL of deionized water to a concentration of 8 mM.

Procedure
1. Serially dilute NADH to the following concentrations: 0, 0.0150625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 mM. Add 200 μL per well to a 96-well plate with three replicates.
2. Measure the absorbance at 340 nm using a microplate reader. Plot a standard curve with the amount of NADH as the abscissa and OD value as the ordinate.
3. Dilute the substrate to 40 mM and set aside.
4. Mix 50 μL of 40 mM substrate with 50 μL of 1 mM NADH, add to the 96-well plate in advance, and set three replicates.
5. Dilute the protein to 0.01 mg/mL. Initiate the reaction by adding 100 μL per well.
6. Zero the microplate reader using the OD value of the blank group, and read the absorbance at 340 nm for 10 min in kinetic mode.
7. Define one enzyme activity unit (U) as the amount that consumes 1 μmol of NADH per minute. Calculate the specific enzyme activity:

Human

HEK293

C-6*His

P09972 (P2-Y364)

230  [NCBI]

Partial

PHSYPALSAEQKKELSDIALRIVAPGKGILAADESVGSMAKRLSQIGVENTEENRRLYRQVLFSADDRVKKCIGGVIFFHETLYQKDDNGVPFVRTIQDKGIVVGIKVDKGVVPLAGTDGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKISERTPSALAILENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHVYLEGTLLKPNMVTPGHACPIKYTPEEIAMATVTALRRTVPPAVPGVTFLSGGQSEEEASFNLNAINRCPLPRPWALTFSYGRALQASALNAWRGQRDNAGAATEEFIKRAEVNGLAAQGKYEGSGEDGGAAAQSLYIANHAY

Approximately 40.0 kDa

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Solution.

1.Supplied as a 0.22 μm filtered solution of 20 mM Tris-HCl, 100 mM NaCl, 50% glycerol, pH 8.0.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.

• [1]. Chang YC, et al. Enrichment of Aldolase C Correlates with Low Non-Mutated IDH1 Expression and Predicts a Favorable Prognosis in Glioblastomas. Cancers (Basel). 2019 Aug 23;11(9). pii: E1238.  [Content Brief]