Log In/Register
ERK Phosphorylation Status Screening Service
Summary
Using our EnzyFluo™ ERK Phosphorylation Assay Kit, we screen for modulators that increase or decrease ERK phosphorylation status in cells. We measure the ERK phosphorylation kinetics, EC50, and IC50 of cells treated with modulators. Testing can be run on a wide variety of cell lines.
Phosphorylation Titration
To determine the time course of ERK phosphorylation, first a titration was run on the concentration of the test compound, here we use phorbol myristate acetate (PMA) as an example. In a sterile, black 96-well plate, PANC-1 cells were plated at a cell density of 10,000 cells per well and allowed to adhere overnight. The next day, the culture medium was removed and replaced with medium dosed with PMA at the following concentrations: 102, 101, 100, 10-1, 10-2, 10-3, 10-4, and 0 ng/mL. The dosed culture medium was prepared from a stock solution of PMA in DMSO so that the final concentration of DMSO in the medium was 1%. Triplicate wells were incubated for 30 minutes with the PMA. The pERK and total protein was then quantified using the ERK Phosphorylation Assay Kit (EERK-100). The pERK values were normalized to the total protein content of the sample.
Phosphorylation Kinetics
After the titration on the test compound concentration, we measure the ERK phosphorylation kinetics for a specific dosage. We will continue to use PMA as an example here; we measure the ERK phosphorylation kinetics for PMA at a concentration of 100 ng/mL. In a sterile, black 96-well plate, PANC-1 cells were plated at a cell density of 10,000 cells per well and allowed to adhere overnight. The next day, the culture medium was removed and replaced with 100 ng/mL PMA dosed medium. The dosed culture medium was prepared from a stock solution of PMA in DMSO so that the final concentration of DMSO in the medium was 1%. Triplicate wells were incubated for 2.5, 5, 10, 20, and 30 minutes with the PMA. The pERK and total protein was then quantified using the ERK Phosphorylation Assay Kit (EERK-100). The pERK values were normalized to the total protein content of the sample.
Inhibitor Titration and IC50 Determination
To determine the IC50 of ERK phosphorylation inhibitors, we run a titration on the concentration of the test compound; here we use the drug Sorafenib as an example. The IC50 of Sorafenib, an ERK phosphorylation inhibitor, was determined for three cell lines: PANC-1, NIH-3T3, and RBL-2H3. The three cell lines were plated in sterile, black 96-well plates at a cell density of 10,000 cells per well and allowed to adhere overnight. The culture medium was aspirated and replaced with culture medium dosed with Sorafenib at the following concentrations: 10-2, 10-4, 10-5, 10-6, 10-7, 10-8, and 10-9 M. The dosed culture medium was prepared from a stock solution of Sorafenib in DMSO so that the final concentration of DMSO in the medium was 1%. Negative control wells received Sorafenib free culture medium with 1% DMSO. All treatment conditions were run in triplicate. The cells were incubated for three hours with the Sorafenib. After the three hour incubation, the Sorafenib medium was aspirated and replaced with 100 ng/mL PMA dosed medium. The cells were incubated with the PMA medium for five minutes. The pERK and total protein was then quantified using the ERK Phosphorylation Assay Kit (EERK-100). The pERK values were normalized to the total protein content of the sample.
IC50 values were calculated using Graphpad Prism. Sorafenib IC50 values were calculated to be 2.1, 11.4 and 11.5 µM respectively, for RBL-2H3, NIH 3T3 and PANC-1 cell lines.
Please email or call 1-510-782-9988 x 2 to request assay service.
Customer Testimonials
"The guys at BioAssay Systems know their science! I had to develop a bioassay for our company and Robert was very quick to respond to keep us up to date. They are knowledgeable and very resourceful. They directed us to one of the bulk suppliers that could give us a really good deal on our active ingredient. They keep very detailed records for our research needs. I would recommend working with BioAssay Systems on your assay development projects!"
"It is a pleasure working together with BioAssay Systems. I brought samples to their Hayward location and received the results in less than 24 hr. The discussion with Robert Z was courteous, open, and with a depth of knowledge. Robert has a PhD from Stanford. Also, Frank Huang, the CEO, had sufficient scientific curiosity to try out our cleanser and provide feedback! This is beyond the call of customer relations. BioAssay Systems is a company with a lot of expertise and a friendly, responsive approach to problem solving. I will enthusiastically continue to work with them to carry out our research."
"I would like to show my appreciation to the Bioassay Systems service team. We are developing an assay to measure the change in lactate concentration of single cell samples using fluorescent microscopy. The BioAssay Systems team was quite helpful in assisting us to optimize their fluorescent L-Lactate assay kit for our unique experimental set-up. The BioAssay Systems team is always friendly and knowledgeable. We know that we can rely on them for fast and professional service."
"While developing biodegradable coatings for Drug Eluting Stents I worked with Bob to develop an accurate, precise and sensitive assay for determining the amount of polymer remaining in tissue. It was a pleasure to work with Bob and his team, they are very thorough, they understand the science very well and they always deliver on time and at the right price. I would highly recommend anyone using BioAssay Systems services in the future."
Previous
Next
