CST BLOG: Lab Expectations

The official blog of Cell Signaling Technology® (CST) where we discuss what to expect from your time at the bench, share tips, tricks, and information.

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Tools for Determining Protein State

Post-translational modifications (PTMs) are a critical regulatory mechanism that allows cells to tightly control protein function. They are reversible, covalent modifications that alter the protein state, such as protein folding, protein-protein interactions, protein-DNA interactions, protein lifespan, protein location, and receptor activation. These behaviors, in turn, control biological processes like enzyme activity, cell cycle, apoptosis, and gene expression.

Some common post-translation modifications include phosphorylation, acetylation, and methylation. A protein can be modified on several different amino acids with different modifications, where different PTM combinations result in different protein behaviors. For example, p53 is modified by as many as 50 individual post-translational modifications. Phosphorylation on p53 on Ser15 and Ser20 can block interactions with MDM2 to promote accumulation and activation of p53 in response to DNA damage, phosphorylation on Ser46 plays a role in apoptosis induction, acetylation on Lys164 induces cell cycle arrest, and acetylation on Lys373 results in hyperphosphorylation of p53 N-terminal residues that in turn enhances the DNA binding ability to promoters for proapoptotic genes.

 

PTMs on other proteins like histones are similarly complex. For example, acetylation of Histone 3 on Lys9 has a dominant role in histone disposition and chromatin assembly while phosphorylation on Ser10, Ser28, and Thr 11 is tightly correlated with chromosome condensation during mitosis and meiosis. Histone 3 methylation, in turn, is primarily associated with transcriptional activation; however, there are scenarios where histone 3 methylation is associated with repression. And that’s just a snapshot of the complexity involved with protein PTMs.

 

Disruptions to the mechanisms that regulate protein PTM states have severe consequences and are found in a wide variety of diseases like cancer, cardiovascular disease, and neurodegenerative diseases. However, we have just started to scratch the surface when it comes to understanding the many different ways proteins can be modified and the implications of those modifications.

 

So how do you even start monitoring the state of proteins in your sample? Western blots are especially useful since you can use highly specific antibodies against the total protein and modification of interest on a particular residue. Don’t have time to find the ideal pair to use? CST has identified duet pairs that are ready for you to start using. Cells can be treated with a modulator or cultured under specific conditions to induce a specific cell state. The specific PTM antibody can then determine the amount of modified protein present compared to the untreated control, and the amount of modified protein present in comparison to the total amount of protein. This can be extremely informative, but only if your PTM specific antibodies do not cross-react with other PTMs on the same protein. Additionally, the PTM specific antibodies need to be sensitive enough to detect even low levels of modified protein.

 

Western blots are also extremely flexible, so you’ll have several options when designing your experiment. Want to multiplex to save on time or sample? Use primary and fluorescently conjugated secondary antibody pairs raised in different species. You could also strip your membrane and re-probe if you want to re-analyze your membrane. Looking to amplify your signal? One option is to use a biotinylated primary antibody, a biotinylated secondary antibody, and streptavidin-HRP to localize more peroxidase molecules to where your protein migrates. Need to immunoprecipitate your protein of interest? Use primary antibodies conjugated to Sepharose® Beads. Or immunoprecipitate your protein of interest with your favorite antibody and Protein A/G agarose or magnetic beads. You even have a lot of options when it comes to selecting which chemiluminescence substrate to use depending on whether you want to maximize signal or signal-to-noise.

 

Cell Signaling Technology® is a company of scientists like you, so we know what you’re looking for in your western blot reagents. Plus, all of our antibodies are validated for specific applications and are specific, sensitive, and reproducible – so you can focus on your research!

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