Signal-Seeker assay kits enable the relevant and simple characterization of the key protein post-modifications (PTMs). In this post, I invite you to look at experimental results obtained with these unique assays developed by Cytoskeleton Inc. for protein Tyrosine phosphorylation and Ubiquitination.
Signal-Seeker technology is based on affinity beads to enrich post-transcriptionally modified proteins from cell or tissue lysates (you might like to look at my previous post in which I describe the Signal-Seeker method). The enriched protein population is then analyzed by standard Western Blotting for identifying the protein of interest with a target-specific primary antibody. This approach enables the convenient analysis of the Tyrosine phosphorylation status or the ubiquitination levels of your protein(s) of interest.
Now let’s take a look at Signal-Seeker kits in action!
Protein Tyrosine Phosphorylation analysis with Signal-Seekers
Tyrosyl Rac1 levels were consistently shown to dip over the first minute of EGF stimulation (repeated 5 times). Interestingly, Rac1 activation, shown in Figure 1B, increased as Rac1 tyrosyl phosphorylation decreased. This observation supports a model that has previously been suggested for kinase regulation of Ras GTPase activity. (1)
Figure 1C shows the total phosphotyrosine signal during EGF stimulation and Figure 1D shows that equal amounts of lysate were used for each IP and activation reaction (10 μl of each lysate was run and analysed by WB using an anti-tubulin antibody (T) and an anti-Rac1 antibody (R) – Arrowhead points to total Rac1 signal from 3% of input lysate).
Signal-Seeker protein Ubiquitination analysis
What about you?
Which PTM are you analyzing? Which protocol are you using, or would you be interested in using the Signal-Seeker technology for your protein of interest? Share your comments, thoughts and questions below!
Subscribe to thematic newsletters on your favourite research topics.