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ACPP/ACP3 His Tag Protein, Human

ACPP/ACP3 His Tag Protein, Human

Catalog Number: UA040045 Brand: UA BIOSCIENCE
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$240 USD
$240 USD
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Product Details

Product Specification


Species Human
Synonyms ACP3,PAP,5'-NT,ACP-3,Prostatic Acid Phosphatase
Accession P15309
Amino Acid Sequence

Lys33-Asp386, with C-terminal His Tag
Expression System HEK293
Molecular Weight

45-50kDa (Reducing)
Purity

＞95% by SDS-PAGE
Endotoxin <0.1EU/μg
Conjugation Unconjugated
Tag His Tag
Physical Appearance Lyophilized powder
Storage Buffer

PBS, 5% Trehalose, pH7.4
Reconstitution

Reconstitute at 0.1-1 mg/ml according to the size in ultrapure water after rapid centrifugation.
Stability & Storage

· 12 months from date of receipt, lyophilized powder stored at -20 to -80℃.

· 3 months, -20 to -80℃ under sterile conditions after reconstitution.

· 1 week, 2 to 8℃ under sterile conditions after reconstitution.

· Please avoid repeated freeze-thaw cycles.

Background

Prostatic acid phosphatase (PAP, or ACPP), also known as prostatic specific acid phosphatase (PSAP), is an enzyme produced by the prostate. As a non-specific phosphomonoesterase, Prostatic acid phosphatase is synthesized and secreted into seminal plasma under androgenic control. The enzyme is a dimer of molecular weight around 100 kDa. Prostatic Acid Phosphatase (ACPP) catalyzes the hydrolysis of a variety of phosphate monoesters, including phosphorylated proteins. The activity optimum of ACPP is in the pH range of 4-6, and the activity is inhibited by L(+)-tartrate. ACPP expression levels are highest in the prostate, with much lower expression in most other tissues. ACPP is a type I integral membrane protein of the plasma membrane and lysosomes, and a secreted form also exists.   The concentration of ACPP is elevated in the circulation of prostate cancer patients, making the enzyme a marker for the progression of prostate cancer. Cellular ACPP has been shown to be a protein tyrosine phosphatase. Protein substrates include the epidermal growth factor receptor and HER-2. Cellular ACPP is considered to function as a tumor suppressor.

Protocol

Assay protocol

Principle: Measured by its ability to cleave a substrate, p-Nitrophenyl phosphate (pNPP).

Materials

  1. Assay Buffer: 50 mM NaOAc, pH 4.5

  2. ACPP His Tag Protein, Human 

  3. Substrate: p-Nitrophenyl phosphate (Sigma-Aldrich, Catalog # 241326)

  4. NaOH, 0.2 M in deionized water

  5. Calibration standard :4-Nitrophenol (Sigma-Aldrich, Catalog #N2765)

  6. 96 Well Clear Plate (BIOFIL, Catalog#011096)

  7. Plate Reader (PerkinElmer)

Produce

  1. Dilute Prostatic Acid Phosphatase Protein to 0.4 μg/mL in Assay Buffer.

  2. Dilute Substrate to 2 mM in Assay Buffer.

  3. Load in a plate 50 μL of dilute Prostatic Acid Phosphatase Protein (From the step 1) and start the reaction by adding 50 μL of 2 mM Substrate to wells. Include a Substrate with Assay Buffer as Blank.

  4. Incubate reaction at room temperature for 5 minutes.

  5. Add 100 μL of 0.2 M NaOH to stop the reaction and develop the color.

  6. Read (top read) absorbance in endpoint mode at 410 nm.

  7. Calculate specific activity.

Specific Activity (pmol/min/µg) =

Adjusted Vmax (OD)x Conversion Factor (pmol/OD)

Incubation time(min) x amount of enzyme (μg)

 

  1. Standard Curve

    1. Dilute Calibration standard to 500 μM in Assay Buffer and prepare serial dilutions.

      1. Add 100 μL of each serially diluted standard and blank (Assay buffer) into appropriate wells of a 96-well, the standard curve has a range of 50000,25000 12500, 6250,3125,1563/781 pmol per well.

        1. Add 100 μL of 0.2 M NaOH to each serially diluted standard blank (Assay buffer).

        2. Read at a wavelength of 410 nm, respectively in endpoint mode.

        3. Linear Regression of 4-Nitrophenol (pmol)(y) –RFU-Blank(x).

 

Picture

SDS-PAGE

2μg (R: reducing conditions, N: non-reducing conditions).