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

Membrane Protein Isolation and Purification With Phase Separation

Posted by The Protein Man on Dec 22, 2014 11:00:00 AM

It is interesting to note that while detergent-solubilized membrane proteins can be purified and concentrated through phase separation (which is a simple, efficient and cost-effective method that can be used for this purpose), most membrane protein scientists do not use this method or even know that such methods exist. To better understand and appreciate how this method works, here are some things you definitely need to know.

Phase Separation: Understanding the Basics

In a nutshell, phase separation (also known as cloud point extraction) refers to the state wherein the micelles in an aqueous solution become immiscible with water and form large aggregates that will separate from the water phase. This condition is usually achieved upon the addition of detergents or when the temperature or salt concentration of the solution is altered.

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Topics: Protein Purification

A Look into the 6XHis Tag and its Uses

Posted by The Protein Man on Oct 20, 2014 9:03:00 AM

The 6xHis tag, also known as polyhistidine tag, His6 tag and/or hexa histidine tag, is an amino acid motif consisting of at least 6 histidine residues fused to the carboxyl (C-) or amino (N-) terminus of a target protein in transfected cells. This tag is most commonly used in the production of recombinant proteins since the string of histidine residues binds to several types of immobilized ions (such as nickel, cobalt and copper) under specific buffer conditions to allow for the simple detection and purification of His-tagged proteins.

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Topics: Protein Purification

Which agarose (sepharose) to choose? 2, 4 or 6%? Crosslinked?

Posted by The Protein Man on Oct 15, 2014 9:00:00 AM

Agarose beads are small spherical beads of agarose gel which are commonly used in gel filtration or molecular size exclusion chromatography and biomolecular purification and immobilization. These beads act as porous gel to filter mixtures of molecules based on their individual sizes. And since these beads are easy to activate, they can also be used to bind biomolecules in a reversible or irreversible manner. In addition, their inert nature and unique internal surface area can also be activated for ligand attachment, making them the ideal basis for various affinity chromatography beads such as protein A and G, and glutathione.

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Topics: Protein Purification, Sample Clean Up

Using Tags in Protein Purification: When Should You Use Them?

Posted by The Protein Man on Sep 8, 2014 9:00:00 AM

Tagging your protein of interest can be extremely useful since it simplifies the purification protocol, improves the yield and solubility of your protein of interest and promotes the proper folding of their fusion partners. Due to their versatility, affinity tags (peptide sequences that are appended to the target protein) are recognized as one of the most powerful tools that can be used for basic biological research and in structural and functional proteomics as well.

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Topics: Protein Purification

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