How to Identify Physiologically Relevant Protein Interactions Using Covalent-Capture HaloTag(R) Technology Information

halotag_blogToday we can see inside the cell and identify protein interactions in their native environment. Many proteins have been characterized in a macromolecular complex, in an individual cell, or in the whole organism. We study proteins in their native environment because they rarely work in isolation. The study of intracellular protein interactions has been challenged by the ability to efficiently capture and preserve protein complexes, especially when attempting to isolate weak or transient interactions. In a recent webinar Rob Chumanov took us through techniques used to study proteins in their native environment and highlighted the most efficient method for studying them based on the HaloTag® covalent tag.

The older generation of protein tags is not ideal for studying protein interactions. These routine protein tags have been adapted for specific narrow applications, such as GFP for live-cell imaging and epitope tags (His, FLAG, and GST) for both fixed-cell imaging and capture of protein:protein interactions. As a consequence, often researchers create multiple protein fusion constructs with different tags in order to optimally characterize protein function. In contrast, HaloTag® technology provides broad flexibility for both imaging and biochemical applications with a single tag that binds rapidly, covalently, and specifically to synthetic small molecule ligands that ultimately determine the functionality of HaloTag®. Continue reading “How to Identify Physiologically Relevant Protein Interactions Using Covalent-Capture HaloTag(R) Technology Information”

One Tag to Rule Them All, One Tag to Find Them, One Tag to Bring Them All and in the Cell or Gel or Column Bind Them

Multiplex Live-Cell Imaging with HaloTag® protein
Typically protein analysis technologies and methods fall into two large buckets: the “biochemical” methods used for detection, purification and studying protein interactions, and the “cell-based” methods for understanding localization and trafficking. For the biochemical analyses, a researcher might employ tools like affinity tags or antibodies; for cell-based studies, a different set of antibodies or fluorescent proteins might be used. The end result is that to study how any one protein functions (where it is, what proteins it interacts with, when it is produced, when it migrates and is degraded) often requires several sets of clones to produce a variety of fusion proteins and a set of antibodies generated against a variety of epitopes.

An ideal protein analysis tool would be flexible enough to lead a researcher through the entire protein analysis workflow, allowing efficient capture and isolation, detection, real-time imaging with high signal and low background at all steps: one tag to find them all, one tag to bind them. In her Promega Webinar, “Accelerating Proteomics Research,” Jacqui Mendez introduced such a protein analysis tool. Continue reading “One Tag to Rule Them All, One Tag to Find Them, One Tag to Bring Them All and in the Cell or Gel or Column Bind Them”

Imperfect Crystal – Inclusion Body

Protein Crystals. Image courtesy of NASA.
Formation of inclusion bodies is one of the most common complications in heterologous protein expression (1). Despite this complication, the E. coli expression system is still highly used for eukaryote protein expression. Is this practice based on knowledge or historic consequence? Continue reading “Imperfect Crystal – Inclusion Body”

6X His Protein Pulldowns: An Alternative to GST

ResearchBlogging.orgPull-down assays probe interactions between a protein of interest that is expressed as fusion protein (e.g.,
(e.g., bait) and the potential interacting partners (prey).

In a pull-down assay one protein partner is expressed as a fusion protein (e.g., bait protein) in E. coli and then immobilized using an affinity ligand specific for the fusion tag. The immobilized
bait protein can then be incubated with the prey protein. The source of the prey protein depends on whether the experiment is designed to confirm an interaction or to identify new interactions. After a series of wash steps, the entire complex can be eluted from the affinity support using SDS-PAGE loading buffer or by competitive analyte elution, then evaluated by SDS-PAGE.

Successful interactions can be detected by Western blotting with specific antibodies to both the prey and bait proteins, or measurement of radioactivity from a [35S] prey protein. bait) and potential interacting partners (prey).

The most commonly used method to generate a bait protein is expression as a fusion protein contain a GST (glutathione-S transferase) tag in E. coli. This is followed by immobilization on particles that contain reduced glutathione, which binds to the GST tag of the fusion protein. The primary advantage of a GST tag is that it can increase the solubility of insoluble or semi-soluble proteins expressed in E. coli.

Among fusion tags, His-tag is the most widely used and has several advantages including: 1) It’s small in size, which renders it less immunogenically active, and often it does not need to be removed from the purified protein for downstream applications; 2) There are a large number of commercial vectors available for expressing His-tagged proteins; 3) The tag may be placed at either the N or C terminus; 4) The interaction of the His-tag does not depend on the tag structure, making it possible to purify otherwise insoluble proteins using denaturing conditions. Continue reading “6X His Protein Pulldowns: An Alternative to GST”