The Protein Man's Blog | A Discussion of Protein Research

SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture)

Quantitative proteomics has been a cutting-edge and demanding field in biological research for the past few decades. With the advancement of technology, MS (Mass spectrometry) based methods have become strikingly helpful tools, which utilize the mass-to-charge ratio of the fragmented compound in proportion with its ionic abundance. MS provides the result as a plot of ionic abundance of a compound versus its mass-to-charge ratio, which subsequently provides information about the nature, possible structure and properties of the compound. This compound could be any protein, posttranslational modification, bioactive molecules etc. The MS responses of proteolytic peptides may discreetly vary owing to their non-uniform physicochemical properties for example, hydrophobicity, dipole moment, size, charge, mass etc. Hence it becomes a strenuous exercise to accurately quantify the changes in the relative abundance of a biomolecule between different test samples in the same MS analysis.  To solve this pitfall, a new modification has been introduced in the general MS protocol known as SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture), which allows insertion of stable isotopes of amino acids into the proteins or peptides. SILAC enables relative and comparable quantification of these molecules from different test samples in the same MS run, which eventually minimize the false variability of the results arising from sample injection and ion suppression contributed by MS instruments in different runs.

Routine H&E (Haemotoxylin and Eosin stains) and special staining comes especially handy when examining tissue structure and cell types and/or when looking for the presence of certain microorganisms in a sample. While both stains are used in histopathology laboratories, H&E stain is more commonly used by pathologists and researchers for investigating underlying cellular and tissue structures.

In all the complex biological processes, the mechanism underlies a synchronized and orchestrated molecular organization, which works through protein-protein interactions. These interactions could be transient, such as catalytic or signal-transduction pathways, requiring a transitory protein-protein association. Alternatively, stable or semi-stable multi-protein complexes are also formed in many biological functions. Hence, in order to identify transient and semi-stable protein associations, chemical cross-linking of proteins came as a useful technique. It involves the formation of chemical covalent bonds between the interacting protein utilizing bifunctional reagents consisting of reactive end groups, which can react with the functional groups of amino acid residues, such as primary amines and sulfhydryls.  The formation of cross-links provides direct and concrete information regarding the identity of the interacting proteins as well as the regions of contact between the proteins.

How do you know which method to use for visualizing proteins after immunodetection? Should you be using the direct detection method or would applying an indirect detection method best serve your purpose? If you’re not sure which method to use, here are some things you definitely need to know.