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

Purify Proteins Fast: How FPLC Speeds the Process

Posted by The Protein Man on Nov 26, 2019 2:30:00 PM

Many researchers use high pressure liquid chromatography (HPLC) to separate and measure concentrations of small molecules. However, this method is relatively ineffective when trying to extract proteins and other biochemicals. Organic solvents used in HPLC place restrictions on these biochemicals, which are unable to function at high temperatures. For this reason, in 1982, Pharmacia developed fast protein liquid chromatography (FPLC) to overcome the weaknesses of the HPLC and institute an effective, quick means to extract and purify biochemicals - especially proteins. 

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Topics: Protein Purification

Protein phosphorylation: Nature’s favorite way of decorating proteins

Posted by The Protein Man on Oct 1, 2019 2:30:00 PM

Part 1: Essentials of protein phosphorylation

American chemist Westheimer asked an interesting question in 1987 in his very famous article published in ‘Science’ the same year, ‘why nature chose phosphates?’. He elegantly approached this problem from a chemists’ perspective. Phosphorus, a group 15 element, is an essential element of biomolecules. Phosphorus and its chemistry pervade cells and living systems, ATP, the ubiquitous currency of energy, is a nucleotide with two phosphates available in high energy phospho anhydride bonds. The backbone of genetic material (DNA or RNA) is made up of phosphates, two adjacent nucleotides are linked by phosphodiester bonds. The negative charge present on phosphodiester linkages in nucleic acids makes them resistant to hydrolysis and therefore very stable in aqueous environments, a key feature required for a molecule to qualify as a genetic material is chemical stability. Other group 15 elements like arsenic don’t make esters that are stable in aqueous conditions at neutral pH.

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Topics: Protein Purification, Protein Labeling

Use of Chaperones in Recombinant Protein Expression

Posted by The Protein Man on Jul 30, 2019 2:30:00 PM

Proteins that form complexes tend to form aggregates during recombinant expression. So the best method is to co-express proteins with suitable partner protein. Chaperones are one of those proteins that help insoluble protein formation. Researchers observed the enhanced solubility of some proteins when co-expressed with chaperones. However, the choice of suitable chaperone for a particular protein is difficult to assume and only with trial and error methods one can achieve a soluble protein. Chaperones act as catalysts that prevent aggregation of nascent chains and unfolding aggregate proteins. Co-expression of recombinant proteins with chaperones help in improved solubility, enhanced specific activity and yield of target proteins.

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Topics: Protein Purification

Protein Purification with IMAC

Posted by The Protein Man on Mar 19, 2019 2:30:00 PM

Protein purification based on fusing peptide affinity tag to recombinant protein is widely preferred due to its ease of use. Immobilized Metal Ion Affinity Chromatography (IMAC) was based on the principle of binding of specific amino acid side chains of a protein to metal ions immobilized on a matrix. Histidine amino acid has high affinity to metal ions, six histidine residues are fused to N-terminal or C-terminal end of a protein during cloning, and the protein obtained could be ~95% pure after passing through metal ion based resin. Protein purification based on metal affinity chromatography is used with all the expression systems including E.coli, Yeast, Insect cell and mammalian cell expression systems. 

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Topics: Protein Purification

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