Kazuya Takes on Far-Western Blotting to Detect Protein Phosphorylation with Wes

"The no-gel, no-transfer, no-membrane features are beneficial for the reproducibility of the assay. I really like this system over traditional Western."

- Kazuya Machida, M.D., Ph.D., Associate Professor, Department of Genetics and Genome Sciences, UConn Health

Kazuya Machida, M.D., Ph.D.

Cancer insights through the SH2 domain

Kazuya Machida is an Associate Professor in the department of Genetics and Genome Sciences at the University of Connecticut Health Center (UConn Health). He studies phosphorylation in tyrosine kinase signaling pathways and the protein-protein interactions that take place when the SH2 domain of one protein binds to a phosphorylated tyrosine residue of the other protein. SH2 domains are often found in adaptor proteins that bind other proteins to trigger the activation or deactivation of downstream proteins in the RTK signaling pathway. Deregulation of RTK signaling pathways lead to a variety of diseases such as cancer and leukemia.

The lab’s research is really extensive. They look at overall phosphorylation patterns and specific phosphorylation of many different proteins in cells and patient tumor tissues. They also compare tyrosine hosphorylation between different cancer types and before and after a drug treatment. All their work is aimed at classifying tumors and understanding downstream effects of different ligand stimulations in the RTK signaling pathway so better cancer treatments can be developed.

Treading carefully with Westerns

The Machida lab monitored tyrosine phosphorylation levels with traditional Western blots to determine whether a protein was present, and Far-Western blots to detect protein-protein interactions. In both methods, proteins were separated by SDS-PAGE and transferred to a membrane. For Far-Western blots, they used a tagged protein domain to probe for the protein of interest instead of a primary antibody. The tagged protein domain interacted with their binding proteins on the blot and that protein-protein complex was then visualized using usual methods like chemiluminescence.

Kazuya used a recombinant GST-tagged SH2 domain to bind denatured proteins with phosphorylated tyrosines and then followed that up with an anti-GST-HRP antibody for chemiluminescent detection. But to get good data, he had to be very careful performing experiments. Everything needed to be done accurately, so a certain skill level was needed to run these blots successfully and reproducibly. One blot would also take up to two days to complete with all the hands-on time needed, so there was a long wait for results. And sometimes he had to load a large amount of precious patient specimens just to get weak signals for interactions that were harder to detect.

Great Far-Western data without the hassle

Wes® automated Kazuya’s blot process so it was a lot easier to get great data compared to traditional blots. The system was easy to customize so he was also able to run his Far-Western experiments by replacing the primary antibody with the GST-tagged SH2. That meant he could use Wes to replace both his traditional and Far-Western blots (Figure 1). Kazuya estimates Wes is about 10X more sensitive than the blots he was running before which lets him reduce the amount of sample loaded, get quantifiable signal for previously hard to detect proteins, and run SH2 domain–ligand interaction experiments more extensively.

Wes also had his runs done in a mere three hours—so he got same-day, fully analyzed results too. This let Kazuya do his research at a much faster pace. He was also able to use Wes’ high molecular weight assay to get better resolution on proteins larger than 200 kDa.

Diving deeper into phosphorylation

Kazuya’s busy developing more assays so he can find better ways to monitor protein phosphorylation. He can’t wait to see what ProteinSimple comes out with next so he can start using it with the hope that his research findings can ultimately prevent a patient from dying from cancer one day.

Figure 1

Figure 1.  Far-Western experiments are more sensitive on Wes compared to traditional Far-Western. (A) EGF-stimulated cell lysates (0.2 or 2 µg/mL) were run on Wes (left) and traditional Western (right) and probed with anti-pTyr antibody (1:50 or 1:500 dilution) as a phosphorylation control. High levels of phosphorylated EGFR and a banding pattern consisting of tyrosinephosphorylated proteins were observed. (B) Far-Western experiments were performed with the same lysates on Wes (left) and traditional Far-Western (right). Samples were probed with Grb2 SH2 (1:10 or 1:100 dilution). Grb2-SH2 binding to p56 and p60 was readily detectable with Wes, indicating specific tyrosine phosphorylation of both proteins. (C) Quantification of signal peaks in the Grb2-SH2 Wes lane view (orange box in B) and electrograph view (orange box in C).

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