Recombinant Human Pepsinogen A Protein, CF

Catalog #: 6155-AS Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
6155-AS-020

R&D Systems Recombinant Proteins and Enzymes

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Recombinant Human Pepsinogen A Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The specific activity is >7,000 pmol/min/μg, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived human Pepsinogen A protein
Met1-Ala388, with a C-terminal 6-His tag

Accession #

P00790

N-terminal Sequence
Analysis

Ile16 and Val63

Predicted Molecular Mass

41 kDa

SDS-PAGE

40-43 kDa, reducing conditions

Product Datasheets

6155-AS

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

6155-AS

Formulation	Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Activation Buffer: 50 mM Sodium Citrate, pH 2.5
Assay Buffer: 50 mM Sodium Citrate, 500 mM NaCl, pH 4.0
Recombinant Human Pepsinogen A (rhPepsinogen A) (Catalog # 6155-AS)
Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002), 2 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhPepsinogen A to 100 µg/mL in Activation Buffer.
Incubate for 15 minutes at room temperature.
Dilute activated rhPepsinogen A to 0.02 µg/mL in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
Load 50 µL of 0.02 µg/mL of rhPepsinogen A into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 µM Substrate.
Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU)
	amount of enzyme (µg)

*Adjusted for Substrate Blank
**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

rhPepsinogen A: 0.001 µg
Substrate: 10 µM

Reconstitution Calculator

Background: Pepsinogen A

Pepsins are aspartic proteases that are synthesized in the gastric mucosa and secreted into the stomach. They are released as zymogens called pepsinogens and then converted to active pepsins by the acidic pH of gastric juices (1). PGA-3, PGA-4, and PGA-5 are isozymogens of human Pepsinogen A, which differ in amino acid sequence by 2-4 residues (2). This recombinant human Pepsinogen A corresponds to PGA-4. Pepsins have optimal activity under conditions of acidic pH and are inhibited by pepstatin. Pepsin A has broad substrate specificity, but preferentially cleaves peptide bonds involving aromatic and aliphatic amino acids.

References