Tag Archives: Size Exclusion Chromatography

Uncovering the Relationship Between Genes and Proteins

What are Genes?

A gene is a basic unit of heredity in a living organism that normally resides in long strands of DNA called chromosomes. Genes are coded instructions that decide what the organism is like, how it behaves in its environment and how it survives. They hold the information to build and maintain an organism’s cells and pass genetic traits to offspring. A gene consists of a long combination of four different nucleotide bases namely adenine, cytosine, guanine and thymine. All living things depend on genes as they specify all proteins and functional RNA chains.

What are Proteins?

Proteins are large, complex molecules that play many critical roles in the body. They are necessary for building the structural components of the human body, such as muscles and organs. Proteins also determine how the organism looks, how well its body metabolises food or fights infection and sometimes even how it behaves. Proteins are chains of chemical building blocks called amino acids. A protein may contain a few amino acids or it could have several thousands. The size of a protein is an important physical characteristic that provides useful information including changes in conformation, aggregation state and denaturation. Protein scientists often use particle size analysers in their studies to discuss protein size or molecular weight.

Archibald Garrod

Archibald Garrod was one of the first scientists to propose that genes controlled the function of proteins. In 1902, he published his observations regarding patients whose urine turned black. This condition known as alkaptonuria happens when there is a buildup of the chemical homogentisate, which causes the darkening of urine. In most situations, excess amounts of amino acid phenylalanine are metabolised by the body. This led Garrod to surmise that the enzyme responsible for its breakdown must be defective in these patients. In addition, since the black urine phenotype was passed from generation to generation in a regular pattern, Garrod reasoned that a gene had to be responsible for the production of the defective enzyme. He attributed a defective enzyme to a defective gene, suggesting a direct link between genes and proteins.

The Relationship Between Genes and Proteins

Most genes contain the information require to make proteins. The journey from gene to protein is one that is complex and controlled within each cell and it consists of two major steps – transcription and translation. Together, these two steps are known as gene expression.

Transcription: Information stored in a gene’s DNA is transferred to a similar molecule called RNA in the cell nucleus. Although both DNA and RNA are made up of a chain of nucleotide bases, they have slightly different chemical properties. The type of RNA that contains the information needed to make protein is called a messenger RNA or mRNA and it carries the message from the DNA out of the nucleus into the cytoplasm.

Translation: This is the second step in the production of proteins and it takes place in the cytoplasm. The mRNA interacts with a specialised complex known as a ribosome that reads the sequence of the mRNA bases. Each sequence has three bases called a codon, which codes for one particular amino acid. A transfer RNA or tRNA assembles the protein, one amino acid at a time. This continues until the ribosome meets a “stop” codon. The characterisation of different proteins can be conducted by Size Exclusion Chromatography as this technique can be used characterise molecular weight, structure and aggregation state.

Learn more about genes and proteins

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The Fundamentals of Liquid Chromatography

Chromatography is the collective term for a set of techniques used to separate mixtures. These techniques include gas chromatography (GC), thin layer chromatography (TLC), Size exclusion Chromatography (SEC), and high performance liquid chromatography (HPLC).

The Two Phases

Chromatography involves passing a mixture dissolved in a “mobile phase” through a “stationary phase”. The mobile phase is usually a liquid or a gas which transports the mixture to be separated through a column or flat sheet which has a solid stationary phase.

Liquid Chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. It can be carried out in either a column or a plane. LC is particularly useful for the separation of ions or molecules that are dissolved in a solvent.

Simple liquid chromatography consists of a column with a fritted bottom that holds a stationary phase in equilibrium with a solvent. Commonly used stationary phases include solids, ionic groups on a resin, liquids on an inert solid support and porous inert particles. The mixture to be separated is loaded onto the top of the column followed by more solvent. The different components in the mixture pass through the column at different rates because of the variations in the partitioning behaviour between the mobile liquid and stationary phases.

Liquid chromatography is more widely used than other methods such as gas chromatography because the samples analysed do not need to be vaporised. Also, the variations in temperature have a negligible effect in liquid chromatography, unlike in other types of chromatography.

High Performance Liquid Chromatography (HPLC)

Present day liquid chromatography that generally utilises tiny packing particles and a fairly high pressure is known as HPLC. It is basically a highly improved form of column chromatography often used by biochemists to separate amino acids and proteins due to their different behaviour in solvents related to the amount of electronic charge of each one.

Instead being allowed to drip through a column under gravity, the solvent is forced through under high pressures of up to 400 atmospheres, making the process much faster. Because smaller particles are used, with their sizes being determined by a particle size analyser, there is greater surface area for interactions between the stationary phase and the molecules flowing past it. This in turn allows for much better separation of the components in the mixture.

There are many advantages of HPLC. For one, it is an automated process that only takes a few minutes to produce results. This is a vast step up from liquid chromatography, which uses gravity instead of a high-speed pump to force components through the densely packed tubing. HPLC produces results that are of a high resolution and are easy to read. Moreover, the tests are easily reproduced via the automated process.

Unfortunately, there are also disadvantages of this technique. It is difficult to detect coelution with HPLC and this may result in inaccurate compound categorisation. The equipment needed to conduct HPLC is also costlier and its operation can be complex.

Thanks to rapid advances in technology, analytical instrumentation such as HPLC are increasing in popularity. For the most part, the efficiency of these techniques outweighs their disadvantages making them a popular choice particularly in the pharmaceutical and medicinal industries.

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