Species Reactivity
Human; Mouse; and hamster
Intended Use
The pERK ELISA Kit is specifically engineered for accurate measurement of total and phosphorylated human, mouse, and hamster ERK 1/2 in cell lysates. Detection of rat ERK 1/2 is expected. The pERK ELISA Kit allows for fast analysis of samples in approximately one hour. All reagents used in a traditional sandwich ELISA are added in solution to a plate followed by a wash step and detection with the TMB colorimetric substrate.
Contents of Kit
This (Total/Phospho) kit includes sufficient reagents to perform 48 wells of the total and 48 wells of the phospho-specific detection for a total of 96 wells.
1. ERK 1/2 (Total) Capture Antibody Reagent – 1 × 1.5 mL
Contains the Capture Antibody Reagent that will be mixed in equal parts to the Detection Antibody Reagent to yield the Antibody Cocktail (ELISA antibody sandwich pair).
2. ERK 1/2 (Total) Detection Antibody Reagent – 1 × 1.5 mL
Contains the Detection Antibody Reagent that will be mixed in equal parts to the Capture Antibody Reagent to yield the Antibody Cocktail (ELISA antibody sandwich pair). The Antibody Cocktail can be prepared by adding an equal volume of Capture Antibody Reagent and Detection Antibody Reagent, and mixing by inversion prior to each experiment.
3. phospho-ERK 1/2 (Thr202/Tyr204, Thr185/Tyr187) Capture Antibody Reagent – 1 × 1.5 mL
4. phospho-ERK 1/2 (Thr202/Tyr204, Thr185/Tyr187) Detection Antibody Reagent – 1 × 1.5mL
5. pERK Assay Plate – 96 well (sealed)
One (1) 96-well strip-well plate specifically designed and manufactured for this assay. The plate is specifically designed to work with this assay and cannot be substituted with other 96-well microplates. Plates should be stored at 2–8°C. Allow plate to equilibrate to room temperature prior to opening the pouch, to minimize condensation from forming in the wells. Unused wells should be stored dry at 2–8°C and used within 1 month of opening the microplate foil bag.
6. Cell Lysis Buffer (5×) – 12 mL
The Cell Lysis Mix is a combination of the Cell Lysis Buffer and Enhancer Solution. The Cell Lysis Buffer (5×) contains a combination of detergents, phosphatase inhibitors, salts, and buffers. Cell Lysis Buffer (5×) is supplemented with Enhancer Solution to yield a versatile Cell Lysis Mix that can be applied to many cells and tissues. Note the dfference in names. Cell Lysis Mix is referred to heavily in the assay protocol.
– The Cell Lysis Mix (5×) is used to lyse cells in the presence of culture medium and is typically used to lyse nonadherent cells.
– The Cell Lysis Mix (1×) is used to lyse cells after the removal of culture medium, and is typically used to lyse adherent cells or nonadherent cells that have been harvested by centrifugation. Cell Lysis Mix (1×) should be used as the diluent for any dilution of cellular lysates that are required.
Note: Supplementing Cell Lysis Mix with extra components (e.g., protease inhibitors, chelating agents, detergents) should be tested on a case-by-case basis for compatibility with pERK ELISA Kit.
7. Enhancer Solution – 1 mL
8. Wash Buffer (10×) – 15 mL
The Wash Buffer, supplied as a 10× concentrate, is used for washing the pERK ELISA Kit microplate. It is a simple mix of buffer, salts, and mild detergent. Alternatively, a PBS, 0.05% (v/v) Tween™ 20 solution may be substituted as a wash solution. If washing wells with a microplate washer, use 3× washes with a 10-second mixing cycle.
9. Positive Control Cell Lysate (lyophilized) – 1 ea
Positive Control Cell Lysate is prepared from various cell types, which have been cultured and prepared to optimize the activation of the intracellular pathway of interest.
– The Positive Control Cell Lysate is intended for use as an assay positive control only, and should not be used for the absolute quantification of a particular protein or phosphorylated target. In combination with negative control wells containing Cell Lysis Mix (1×) only, the Positive Control Cell Lysate can be used to give an indication of the expected signal range for a given assay.
– The Positive Control Cell Lysate controls are supplied lyophilized, and should be reconstituted with 250 μL of reagent grade dd H2O. If required, Positive Control Cell Lysate can be further diluted with Cell Lysis Mix (1×), and frozen at less than –20°C in aliquots for subsequent use.
10. Detection Reagent (TMB Substrate) – 12 mL
The emission filter should be in the range of 450 nm, with bandwidths ≤30 nm. The signal in the wells should be developed for around 15 minutes. Best results will be obtained if the microplates are developed in the dark (e.g., by covering the microplate with foil). It is recommended to protect the plate from light while undergoing development.
11. Stop Solution – 12 mL
The Stop Solution is used for stopping HRP-mediated colorimetric conversion. When added to the wells, the HRP enzyme activity stops and the detection reagent turns from blue to yellow with deeper yellow indicating a higher concentration of target over a lighter development. The plate should be read immediately after the addition of the stop solution.
12. Plate Seal – 1 ea
Storage
Store kit components at the temperatures indicated on the labels. When handled as described below, the kit is stable for 6 months from date of receipt. Store all reagents at 2–8°C. Do NOT freeze the kits.
Opened/reconstituted kit reagents:
1. Capture Antibody Reagent: Store at 2–8°C after opening, until expiry date.
2. Detection Antibody Reagent: Store at 2–8°C after opening, until expiry date.
3. Cell Lysis Buffer (5×): Store at 2–8°C after opening, until expiry date.
4. Enhancer Solution: Store at 2–8°C after opening, until expiry date.
5. Wash Buffer (10×): Store at 2–8°C after opening, until expiry date.
6. Detection Reagent: Store at 2–8°C after opening, until expiry date.
7. Stop Solution: Store at 2–8°C after opening, until expiry date.
8. pERK Assay Microplate: Return unused wells to foil pouch with dessicant pack, and seal with tape. May be stored at 2–8°C for up to 1 month after opening.
9. Positive Control Cell Lysate: Aliquot and store at <-20°C for up to 1 month.
General Description
Extracellular signal-regulated kinase (ERK) is the founding member and a key component of the classical Mitogen Activated Protein Kinase (MAPK) pathway. ERK1 (p44, MAPK3) and ERK2 (p42, MAPK2) are both activated via the MAPK/ERK pathway, which is downstream of various Receptor Tyrosine Kinases (RTKs) or GPCRs. In the classical MAPK pathway, ligand binding induces the activation of an RTK that initiates a signaling cascade that results in activation of the GTPase Ras and the Ser/Thr kinase Raf (MAPKKK). Raf then binds to and activates MEK (MAPKK) via phosphorylation. MEK, a Ser/Thr and Tyr kinase, activates ERK by phosphorylation of its TxY motifs, namely Thr202/Tyr204 and Thr185/Tyr187 of ERK1 and ERK2, respectively. Both phosphorylation sites are required for ERK activation and no known mutations exist that cause constitutive ERK 1/2 activation. As such, detection of ERK 1/2 phosphorylation is frequently used to assess its activation, as well as that of MEK, a popular drug target. Once activated, ERK phosphorylates a variety of proteins that regulate cellular processes such as cell division, proliferation, survival, differentiation, apoptosis, motility, and metabolism. Due to its established critical role in these processes, the MAPK signaling pathway is a frequent target for oncology-related drug development.
Standard Curve
Confluent HeLa cells were serum starved overnight, then stimulated with EGF for 10 min at the indicated concentrations. Cells were analyzed for ERK 1/2 (pT202/Tyr204) phosphorylation or total ERK 1/2 protein levels using the pERK ELISA Kit, respectively.
Citations
Publication ()
Have you cited DEIA4043 in a publication?
Let us know and earn a reward for your research.
COA
Loading ......
