Alkaline Phosphatase/ALPP Protein, Human (HEK293, C-His)

Alkaline Phosphatase/ALPP Proteinas, an enzyme, effectively hydrolyzes various phosphate compounds. Alkaline Phosphatase/ALPP Protein, Human (HEK293, C-His) is the recombinant human-derived Alkaline Phosphatase/ALPP protein, expressed by HEK293 , with C-6*His labeled tag.

Alkaline phosphatase (ALP), encoded by the ALPP gene, is an enzyme known for its efficient hydrolysis of various phosphate compounds under alkaline conditions. This protein plays a crucial role in maintaining phosphate homeostasis and is involved in diverse physiological processes. ALP is notably expressed by osteoblasts in bone tissue, where it is essential for mineralization during bone formation. Moreover, ALP serves as a valuable biomarker, particularly in assessing liver and bone health, with elevated serum levels indicative of certain disorders.

Immobilized  Human ALPP at 1 μg/mL (100 μL/well) can bind Anti-ALPP Antibody, The ED₅₀ for  this effect is 3.432 ng/mL.

Materials
Alkaline Phosphatase/ALPP Protein, Human (HEK293, C-His) (HY-P7487A)
Wash buffer: 1X PBST
Blocking buffer: 1% BSA in 1X PBS-T
TMB Substrate Solution: Mix Substrate Solution A and B at a 1:1 ratio immediately before use
Stop Solution: 2 M H₂SO₄

Procedure
1. Coating: Dilute the test protein to 1 μg/mL in 1X PBS, add 100 μL/well, and incubate overnight at 4°C. 2. Washing: Wash the plate with wash buffer (260 μL/well) , once, and pat dry.
3. Blocking: Add 150 μL/well of blocking buffer (1% BSA in 1X PBST) , incubate at 25°C with shaking at 450 rpm for 4 hours.
4. Dilute Primary Antibody: Dilute the antibody to concentrations of 0, 0.01, 0.1, 1, 5, 10, 50, 100, 500, 1000, 5000, 10000, 50000 ng/mL.
5. Add Primary Antibody: Add 100 μL/well, incubate at 25°C with shaking at 450 rpm for 2 hours. 6. Washing: Wash the plate with wash buffer (260 μL/well) , five times, with each wash lasting 1 minute, then pat dry.
7. Add Secondary Antibody: Add 100 μL/well, incubate at 25°C with shaking at 450 rpm for 1 hour.
8. Washing: Wash the plate with wash buffer (260 μL/well) , three times, with each wash lasting 1 minute, then pat dry.
9. Add TMB Substrate Solution: Add 100 μL/well, incubate at room temperature in the dark for 15 minutes.
10. Stop Reaction: Add 100 μL of 2 M H₂SO₄ per well.
11. Read Plate: Measure absorbance at 450 nm.
12. Using GraphPad Prism software, plot a curve with OD values on the vertical axis and primary antibody concentration on the horizontal axis to calculate the ED₅₀ value.

Human

HEK293

C-6*His

P05187 (I23-D506)

250  [NCBI]

Full Length of Mature Protein

IIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTD

approximately 62.5 kDa

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

Lyophilized powder.

Lyophilized from a 0.2 μm filtered solution of 20 mM PB, 150 mM NaCl, 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.