Recombinant Human PKLR Protein, CF

R&D Systems | Catalog # 8569-PK

Pyruvate Kinase Isozymes L/R
R&D Systems
100% Guarantee

Key Product Details

  • R&D Systems E. coli-derived Recombinant Human PKLR Protein (8569-PK)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P30613

Applications

Enzyme Activity
View all PKLR Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human PKLR protein
Leu47-Ser574, with N-terminal Met and 6-His tag

Purity

>85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

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

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

58 kDa

SDS-PAGE

51-60 kDa, reducing conditions

Activity

Measured by its ability to transfer phosphate from phospho(enol)pyruvic acid monosodium salt hydrate (PEP) to adenosine 5'-diphosphate sodium salt (ADP).
The specific activity is >25,000 pmol/min/ug, as measured under the described conditions.

Reviewed Applications

Read 1 review rated 5 using 8569-PK in the following applications:

Formulation, Preparation, and Storage

8569-PK
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and DTT.
Shipping The product is shipped with dry ice or equivalent. 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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: PKLR

Pyruvate kinases (PK) are glycolytic enzymes that catalyze the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP, the final step in the glycolysis pathway (1, 2). There are 4 isozymes of pyruvate kinase in mammals: L, R, M1 and M2. L type is the major isozyme in the liver, R is found in red cells, M1 is the main form in muscle, heart and brain, and M2 is found in early fetal tissues. R and L types are encoded in a same gene and caused by different splicing (3). The 33 amino acids from the N-terminus in the R type are placed with Met and Glu in the L type. PKLR is allosterically activated by fructose
1,6-bisphosphate (4). Mutation of PKLR is a frequent cause of hereditary non-spherocytic hemolytic anemia (5-8).

References

  1. Kanno, H. et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:8218.
  2. Tani, K. et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:1792.
  3. Kanno, H. et al. (1992) Biochem. Biophys. Res. Commun. 188:516.
  4. Valentini G. et al. (2002) J. Biol. Chem. 277:23807.
  5. Baronciani, L. and Beutler, E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:4324.
  6. Beutler E. and Baronciani L. (1996) Hum. Mutat. 7:1.
  7. Baronciani, L. et al. (1996) Blood Cells Mol. Dis. 22:259.
  8. Van Wijk, R. et al. (2009) Hum. Mutat. 30:446.

Long Name

Pyruvate Kinase, Liver And RBC

Alternate Names

PK1, PKL, Pyruvate Kinase 1, RPK

Entrez Gene IDs

5313 (Human); 18770 (Mouse)

Gene Symbol

PKLR

UniProt

P30613

Additional PKLR Products

Product Documents for Recombinant Human PKLR Protein, CF

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

Download SDS

Product Specific Notices for Recombinant Human PKLR Protein, CF

For research use only

Citations for Recombinant Human PKLR Protein, CF

Customer Reviews for Recombinant Human PKLR Protein, CF (1)

5 out of 5
1 Customer Rating
5 Stars
100%
4 Stars
0%
3 Stars
0%
2 Stars
0%
1 Stars
0%

Have you used Recombinant Human PKLR Protein, CF?

Submit a review and receive an Amazon gift card!

$25/€18/£15/$25CAN/¥2500 Yen for a review with an image

$10/€7/£6/$10CAN/¥1110 Yen for a review without an image

Submit a review
Amazon Gift Card

Customer Images

Showing  1 - 1 of 1 review Showing All
Filter By:
Clear Filters X
  • Recombinant Human PKLR Protein, CF
    Name: Anonymous
    Application: Enzymatic activity in vitro
    Verified Customer | Posted 01/27/2024
    Recombinant Human PKLR Protein, CF 8569-PK

There are no reviews that match your criteria.

Showing  1 - 1 of 1 review Showing All

Protocols

View specific protocols for Recombinant Human PKLR Protein, CF (8569-PK):

Materials
  • Assay Buffer: 0.1 M MES, 5 mM MgCl2, 20 mM KCl, pH 6.5
  • Recombinant Human PKLR (rhPKLR) (Catalog # 8569-PK)
  • Activator: D-Fructose 1,6-bisphosphate trisodium salt hyrate (Sigma, Catalog # F6803), 50 mM stock in deionized water
  • Adenosine 5’-diphosphate sodium salt (ADP) (Sigma, Catalog # A2754), 200 mM stock in diH2O
  • Phospho(enol)pyruvic acid monosodium salt hydrate (PEP) (Sigma, Catalog # P0564), 50 mM stock in deionized water
  • Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
  • Kinase Glo® Plus Luminescent Kinase Assay (Promega, Catalog # V3771)
  • 96-well Solid White Plate (Corning Life Sciences, Catalog # 3912)
  • Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
  1. Follow the Kinase Glo® Plus Luminescent Kinase Assay kit directions for preparing Kinase Glo® Plus Reagent. Use the reagent immediately or freeze aliquots at -20°C for future use. (Note: This assay measures an increasing luminescent signal, which is opposite of the traditional use of the kit).
  2. Dilute rhPKLR to 0.25 µg/mL in Assay Buffer.
  3. Dilute Activator to 0.133 mM in Assay Buffer.
  4. Stimulate enzyme by combining 40 µL of 0.25 µg/mL rhPKLR and 60 µL of 0.133 mM Activator. Replace enzyme with Assay Buffer for a Control.
  5. Incubate for 5 minutes at room temperature.
  6. Prepare a substrate mixture of 5 mM ADP and 4 mM PEP in Assay Buffer.
  7. Add 100 µL of the substrate mixture to incubated rhPKLR and Control. Mix briefly.
  8. Incubate all at room temperature for 15 minutes.
  9. Dilute ATP to 100 µM in Assay Buffer.  This is the first point of the standard curve. Complete the curve by performing six two-fold dilutions of 100 µM ATP in Assay Buffer. The standard curve has a range of 0.078 to 5.0 nmol per well.
  10. Load 50 µL of each dilution of the standard curve in triplicate into a solid white plate. Include a curve blank containing 50 μL of Assay Buffer.
  11. After incubation (step 8), heat reactions and control at 90-100 °C for 1 minute to stop the reaction. Cool on ice and centrifuge briefly to collect any condensation.
  12. Load 50 µL of each reaction and control in triplicate into the same plate containing the standard curve.
  13. Add 50 µL of Kinase Glo® Plus Reagent to all wells used, including standard curve.
  14. Incubate at room temperature for 10 minutes in the dark.
  15. Read plate in Luminescence endpoint mode.
  16. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adj. Luminescence* (RLU) x conversion factor** (nmol/RLU) x 1000 pmol/nmol
Incubation time (min) x amount of enzyme (µg)


*Adjusted for Control.
**Derived from the ATP standard curve using linear fitting and adjusted for Curve Blank.

Per Well:
  • rhPKLR: 0.0025 µg
  • ADP: 1.25 mM
  • PEP: 1.0 mM
  • Activator: 20 µM

FAQs

No product specific FAQs exist for this product.

View all FAQs for Proteins and Enzymes