Carbonic Anhydrase 13 Protein, Human (His)

Carbonic Anhydrase 13 Protein, Human (His) expresses in E. coli with a His tag at the N-terminus. Human Carbonic Anhydrase 13, a member of the human carbonic anhydrase (hCA), can control pH and ion balance regulation^[1].

The cytosolic isoform XIII is a recently discovered member of the human carbonic anhydrase (hCA, EC 4.2.1.1) family. It is selectively expressed among other tissues in the reproductive organs, where it may control pH and ion balance regulation, ensuring thus proper fertilization conditions^[1].

Measured by its esterase activity. The specific activity is 186.42 pmol/min/µg that incubate at room temperature in kinetic mode for 5 minutes.

Materials
Assay buffer: 12.5 mM Tris, 75 mM NaCl, pH 7.5
Carbonic Anhydrase 13 Protein, Human (His) (HY-P7726)
Substrate: 4-Nitrophenyl Acetate, dissolved in 2 mM DMSO
Standard: 4-Nitrophenol

Procedure
1.Standard Preparation: Dilute 4-Nitrophenol in assay buffer to concentrations of 100、50、25、12.5、6.25、3.125、1.5625、0 μmol/L. Dispense 100 μL of each standard concentration into a 96-well plate. Read at 400 nm wavelength (top read) for 5 minutes in kinetic mode. Plot the standard curve with standard concentration on the y-axis and measured OD on the x-axis to derive the standard curve equation.
2. Dilute the protein sample to 100 μg/mL using assay buffer.
3. Dilute the substrate to 2 mM using assay buffer.
4. Add 50 μL of the test protein to a 96-well plate and add 50 μL of 2 μM substrate to initiate the reaction. Add 50 μL of assay buffer and 50 μL of 2 μM substrate to the blank wells.
5. Read at 400 nm wavelength (top read) for 5 minutes in kinetic mode.
6. Calculate enzyme activity:

Human

E. coli

C-6*His

Q8N1Q1 (M1-F261)

377677  [NCBI]

Partial

MSRLSWGYREHNGPIHWKEFFPIADGDQQSPIEIKTKEVKYDSSLRPLSIKYDPSSAKIISNSGHSFNVDFDDTENKSVLRGGPLTGSYRLRQVHLHWGSADDHGSEHIVDGVSYAAELHVVHWNSDKYPSFVEAAHEPDGLAVLGVFLQIGEPNSQLQKITDTLDSIKEKGKQTRFTNFDLLSLLPPSWDYWTYPGSLTVPPLLESVTWIVLKQPINISSQQLAKFRSLLCTAEGEAAAFLVSNHRPPQPLKGRKVRASF

Approximately 32.0 kDa

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

Solution.

Supplied as a 0.22 μm filtered solution of 20 mM Tris-HCl, 150 mM NaCl, pH 7.5.
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.

N/A.

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]. Anna Di Fiore, et al. Crystal structure of human carbonic anhydrase XIII and its complex with the inhibitor acetazolamide. Proteins. 2009 Jan;74(1):164-75.  [Content Brief]