Mastering Post-Translational Modifications

Post-translational modifications (PTMs) play a key role in dynamic cellular processes, regulating gene expression, protein activity, localization, and degradation, as well as protein interaction. Modification-specific antibodies offer a versatile tool for the characterization of post-translational modifications. Learn how you can choose the best high-affinity, high-specificity antibody for your PTM detection needs.

At Rockland, scientists have developed proprietary methods for the development of highly specific PTM antibodies that can be used in a wide range of in vitro and in vivo studies of a modified protein, some of which are not easily performed by other approaches, such as mass spectrometry (MS).

Antibody Selection Tips for PTMs

1 Preparation

From an antibody production point of view, the differences between modified proteins can be quite small. Peptide design and immunogen quality are critical to the generation of a specific immune response to ensure the production of high-quality antibodies.

2 Production

Antibodies against PTMs are generated using a short, specific region of the protein, largely eliminating the issue of specificity seen with antibodies generated using large constructs as immunogens. However, it is critical that the antibody be tested against established positive and negative controls to ensure specificity for the modification. Polyclonal antibodies can be immunodepleted during production if the sample contains antibodies that recognize other PTMs.

3 Validation

Dot blot assays and ELISAs can be used to assess both antibody specificity and sensitivity. Keep in mind that, in addition to being specific for the required modification, the antibody must be validated for the application of choice using appropriate positive and negative controls.

Common PTMs & Their Functions

Phosphorylation

Protein phosphorylation is controlled by kinases and phosphatases, and plays a significant role in a wide range of cellular processes, including cell growth and proliferation, metabolism, physiological regulation, and cell signaling.

SUMOylation

Sumoylation involves the addition of small ubiquitin-like modifiers (SUMOs) that enhance stability or modulate the subcellular compartmentalization of proteins. It has been implicated in various cellular processes, such as nuclear transport, signal transduction, stress response, and cell cycle progression.

Glycosylation

Attachment of glycans to proteins is critical for protein folding, stability, targeting, and binding. Five types of glycosylation are observed: N- and O-linked glycosylation, C-linked mannosylation, glypiation, and phospho-serine glycosylation.

Acetylation

Acetylation, or the addition of an acetyl group at lysine residues is a major posttranslational modification for histones, regulating gene expression and metabolism.

Methylation

Protein methylation is a reversible process by which methyl groups are added to arginine or lysine residues, mediated by peptidylarginine or lysine methyltransferases.

Ubiquitination

Ubiquitination is an essential cellular process that tags abnormal, foreign, and improperly folded proteins, targeting them for degradation by the 26S proteasome.

Palmitoylation

S-Palmitoylation involves the lipid modification of cystine residues with palmitic acid. This modification plays a role in protein localization, stability, subcellular trafficking, and protein-protein interaction.