The Ras-Raf-MEK-ERK Pathway is an important cell signalling route with many implications for cancer biology and therapeutic development. Dis-regulation of some of this pathway’s proteins expression and phosphorylation status are observed in about one-third of all human cancers. Access to specific tools to study this pathway is essential to a better understanding of its role in cancer for novel drug development. If you are working on this topic, you’ll be interested in taking a look at the range of reagents on offer for a wide variety of applications.
Cell Signalling: evaluate the phosphorylation status of MEK and ERK
As shown in the illustration below, the MEK and ERK protein families act early in the MAPK/ERK pathway cascade of events. MEK1/MEK2 are activated by Raf through the phosphorylation of two serine sites: S218/S222 and S222/S226 respectively. Activated MEK enzymes then catalyze the phosphorylation of human ERK1 and ERK2 at 2 distinct activation residues: ERK Tyr204/187 respectively, followed by ERK Thr202/185. The phosphorylation of both the tyrosine and threonine is required for enzyme activation.
Therefore, the need for tools to evaluate the phosphorylation status of MEK and ERK appears essential in order to study this cell signalling route. This post will help you choose from a selection of reagents sorted by application.
If you’re working in western blot or ELISA, you may be interested in the new antibodies from Rockland recently released. I have made a selection for you of mouse monoclonal antibodies anti-MEK1 and MEK-2, as well as Phospho-specific antibodies anti-MEK1 pS222 and anti-MEK2 S226.
Available from Assay Biotech, an anti-p MEK2 directed against Thr394 site is guaranteed in Western Blot, IHC, and ELISA and only detects the phosphorylated form of the protein. They have also developed anti-phospho MEK1 for the 298, 286 and 291 phosphorylation sites.
MEK1 and MEK2 double mutant human recombinant proteins offer convenient negative control protein for immunoassays using antibodies targeting the critical MEK1 phosphorylated S218 or S222 residues, and the MEK2 S222A and S226A respectively, with alanine substitution at both sites. Similar tools are available for ERK1 and 2.
If you would rather go for a Sandwich ELISA format to quantify the phosphorylated form of your protein in cell lysates, then RayBiotech has developed a whole range of Phospho ELISA Kits for ERK and MEK proteins. These kits are advantageous if you want to simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blot analysis.
If your aim is rather to look at the ratio of phosphorylated protein versus total protein, to screen the effect of activators or inhibitors in your cells, the ERK cell based assays will address your need.
If on the other hand you’re aiming at profiling the key proteins of the MAP Kinase signaling cascades, a semi-quantitative method allowing you to profile more targets may be more suited to what you want to do. You may wish to consider either the MAPK Pathway Phospho Antibody Array (185 phospho-specific antibodies) or the MAPK signalling array (63 target proteins). Each package contains two identical array slides for analyzing two samples, such as an untreated sample and a treatment sample.
Finally, if you are looking for biologically active proteins, BPS offer a range of fully tested proteins for MEK and ERK.
What about other cell signalling pathways?
You might be interested in taking a look at some of the other topics we’ve covered in this area, such as MAP Kinase cell signalling and the ELISA tests you can use. Or also, the JAK/STAT pathway and some of the best tools for studying it, such as ELISA tests, antibody arrays and antibodies.
So much to choose from… if your head is spinning by now or if you have any questions about the best tools to study pathways and cell signalling, don’t hesitate to get in touch by leaving a comment below. I’ll be pleased to get back to you!