Recombinant Human Active ERK1 Protein, CF Summary
Product Specifications
Analysis
Product Datasheets
Carrier Free
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.
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.
1879-KS
Formulation | Supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 mM DTT, 0.1 mM PMSF and 25% glycerol. |
Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
Stability & Storage: | This product is stable at ≤ ‑70°C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles. |
Assay Procedure
- Active Kinase - Active ERK1 (0.1 μg/μL) diluted with Kinase Dilution Buffer and assayed as outlined in Sample Activity Plot. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
- Kinase Assay Buffer III - 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2, and 0.5 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 250 μM.
- Kinase Dilution Buffer IX (1x) - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold dilution) with cold distilled water. Add fresh DTT prior to use to a final concentration of 50 μM.
- ADP-GloTM Kinase Assay Kit - 10 mM ATP Solution, 10 mM ADP Solution, ADP-GloTM Reagent, Kinase Detection Reagent.
- Substrate - Myeline basic protein (MBP) diluted in 100 mM MOPS buffer (pH 6.5) to a final concentration of 0.5 mg/mL.
- Thaw the Active ERK1, Kinase Assay Buffer III (5x), and Substrate on ice. Prepare a 15 μL enzyme dilution using Kinase Dilution Buffer IX (1x), at the desired concentration, in a pre-chilled 96-well plate.
- Prepare a substrate/ATP mixture as follows (25 μM ATP example)
a. 10 mM ATP Solution: 1 μL
b. Kinase Assay Buffer III (5x): 79 μL
c. Substrate at 0.5 mg/mL: 80 μL - Transfer the following reaction components prepared in Step 1 and 2 to a 384-well opaque plate bringing the reaction volume up to 5 μL:
a. 3 μL of diluted Active ERK1
b. 2 μL of Substrate/ATP mix as prepared in the Step 2. This initiates the reaction. - Set up the blank control as outlined in step 2, excluding the addition of the kinase. Replace the kinase with an equal volume of Kinase Dilution Buffer IX (1x).
- Incubate at ambient temperature for 40 minutes.
- After the 40 minute incubation period, terminate the reaction and deplete the remaining ATP by adding 5 μL of ADP-Glo Reagent. Spin down and shake the 384-well plate. Then incubate the reaction mixture for another 40 minutes at ambient temperature.
- Add 10 μL of the Kinase Detection Reagent to the 384-well plate and incubate the reaction mixture for another 30 minutes at ambient temperature
- Read the 384-well reaction plate using the Luminescence Module Protocol on a GloMax®-Multi Microplate Multimode Reader.
- Determine the corrected activity (RLU) by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below.
Calculation of Specific Activity of ADP (RLU/pmol)
From ATP-ADP conversion curve, determine RLU/pmol of ADP
Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)]
Scientific Data
Reconstitution Calculator
Background: ERK1
ERK1 is a protein Serine/Threonine kinase that is a member of the extracellular signal-regulated kinases (ERKs) which are activated in response to numerous growth factors and cytokines (1). Activation of ERK1 requires both tyrosine and threonine phosphorylation that is mediated by MEK. ERK1 is ubiquitously distributed in tissues with the highest expression in heart, brain, and spinal cord. Activated ERK1 translocates into the nucleus where it phosphorylates various transcription factors.
Citations for Recombinant Human Active ERK1 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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ERK1/2 inhibits Cullin 3/SPOP-mediated PrLZ ubiquitination and degradation to modulate prostate cancer progression
Authors: Y Fan, T Hou, W Dan, Y Zhu, B Liu, Y Wei, Z Wang, Y Gao, J Zeng, L Li
Cell Death and Differentiation, 2022-02-22;0(0):.
Species: Human
Sample Types: Protein
Applications: Bioassay -
Phosphatase-coupled universal kinase assay and kinetics for first-order-rate coupling reaction.
PLoS ONE, 2011-08-11;6(8):e23172.
Applications: Enzyme Assay
FAQs
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What is the activation protocol for this enzyme?
This enzyme is supplied in its activated form, so no activation step is required.
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