BACE1 Protein, Human (HEK293, N-His)

BACE1 protein is crucial in proteolytic processing of amyloid precursor protein (APP), initiating cleavage at the N-terminus of the A-beta peptide sequence. This process results in the extracellular release of beta-cleaved soluble APP and a cell-associated C-terminal fragment. BACE1's cleavage activity extends to CHL1, demonstrating its versatility in substrate processing. BACE1 Protein, Human (HEK293, N-His) is the recombinant human-derived BACE1 protein, expressed by HEK293 , with N-6*His labeled tag.

The BACE1 protein plays a pivotal role in the proteolytic processing of the amyloid precursor protein (APP). It initiates cleavage at the N-terminus of the A-beta peptide sequence, specifically between residues 671 and 672 of APP, resulting in the production and subsequent extracellular release of beta-cleaved soluble APP. Simultaneously, BACE1 generates a corresponding cell-associated C-terminal fragment, subsequently released by gamma-secretase. In addition to its primary target, BACE1 also exhibits cleavage activity on CHL1, as observed through its similarity in cleaving this substrate.

Measured by its ability to cleave a fluorogenic peptide substrate, Mca-SEVNLDAEFRK(Dpn)RR-NH2 (HY-P1859). Read at excitation and emission wavelengths of 320 nm and 405 nm. The specific activity is >3.5 pmoL/min/µg.

Materials
Assay buffer: 0.1 M Sodium Acetate, pH 4.0
Test protein: BACE1 Protein, Human (HEK293, N-His) (HY-P700606)
Substrate: MCA-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys(DPN)-Arg-Arg-NH2 (HY-P1859)

Procedure
1. Reconstitute the protein with sterile water to a concentration of 200 μg/mL, then dilute human BACE1 protein to 20 μg/mL using assay buffer.
2. Dilute the substrate to 20 μM using the assay buffer.
3. Experimental group: 50 μL BACE1 + 50 μL, 20 μM substrate;
Control group: 50 μL assay buffer + 50 μL, 20 μM substrate.
4. In kinetic mode, read for 5 minutes at an excitation wavelength of 320 nm and an emission wavelength of 405 nm.
5. Calculate the specific activity:

Human

HEK293

N-6*His

P56817-1 (T22-T457)

23621

Extracellular Domain

TQHGIRLPLRSGLGGAPLGLRLPRETDEEPEEPGRRGSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLSSTYRDLRKGVYVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWEGILGLAYAEIARPDDSLEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRPVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSACHVHDEFRTAAVEGPFVTLDMEDCGYNIPQTDESTLMT

Approximately 55-75 kDa, based on SDS-PAGE under reducing conditions, due to the glycosylation.

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

Lyophilized powder

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.