Purification

Introduction

Purification (sometimes called isolation or extraction) refers to the physical separation of a target molecule such as nucleic acids (DNA / RNA) or proteins from a sample. Purification techniques are a starting point for most molecular biological experiments, as nucleic acids or proteins are the starting material for many research projects. Though colloquially they are assumed to be the same, there are minor differences, which are explained below.

This knowledge page covers:

  • DNA Purification (inlcuding genomic DNA purification and plasmid purification)

  • RNA Purification

  • Protein Purification

There is also an example of how purification kits can be easily compared when using ZAGENO.

The Purification Process

The process of purification is a combination of physical and chemical methods, which can vary depending on the target molecule and the sample type. For example, genomic DNA extraction procedures differ from mRNA or plasmid extraction procedures. Also the extraction of genomic DNA can diverge in technique according to the sample type, such as blood or tissue . In general, the steps of purification involve lysis, extraction, and cleanup .

For post-purification clean-up procedures, you can read and compare products under that subcategory.

DNA Purification

DNA Purification is the process of removing impurities from isolated DNA material. Originally performed in 1869 by Swiss scientist Friedrich Miescher, DNA purification has become a key procedure for a majority of molecular biological experiments. Whether it is PCR, sequencing, or genotyping, DNA purification is an integral first step, as contamination may hinder downstream processing.

To complicate things further, the expressions: DNA purification, DNA extraction, and DNA isolation are often used interchangeably for the same processes. However, there are some slight differences. While DNA isolation aims to get as much of the DNA out of your sample as possible, DNA purification is done to reduce, or even eliminate, the contamination of the isolated DNA. Extraction is just one specific way to achieve isolation and purification.

The process of purification varies depending on sample type and extraction method (or downstream applications). For example, plasmid DNA purification has different protocols compared to genomic DNA purification. Nonetheless, the overlying principles remain the same.

Firstly, cells are broken down to expose their DNA. Secondly, the removal of other material such as membrane lipids, proteins, and RNA (done separately). Lastly, the purification of DNA itself. In terms of extraction methods, silica columns or magnetic beads give the best reproducibility while ethanol precipitation is well suited for extractions from large input volumes.

Cell lysis is the initial step for DNA purification. The cellular structure needs to be broken down to expose the underlying DNA in the cell nucleus. This is done with chaotropic salts, detergents or alkaline denaturation for plasmid DNA purification.

  • Chaotropic salts: Common chaotropic agents are phenol, ethanol, guanidine hydrochloride, urea, and lithium perchlorate. These substances denature proteins and nucleic acids, but more importantly, set the stage for the binding of DNA to a silica substance: the most common method for DNA extraction.

  • Detergents: Aid in further degradation of protein and cellular structures. Detergents are used in DNA extraction to degrade the cell membrane and nuclear envelope, allowing DNA to be released.

  • Enzymes: Further help the degradation of protein and RNA. This is commonly achieved with Proteinase K (a broad spectrum serine protease), and Ribonuclease (RNase). Proteinase K is highly suitable for DNA extraction, as it is able to function while in the presence of the other chemicals in the lysis buffer. RNase is an enzyme which catalyzes the breakdown of RNA, which can contaminate downstream applications.

Once cell lysis has occurred, the next step is the removal of contaminants such as carbohydrates and other macromolecules that are present in the cell. The removal process is often carried out through centrifugation and subsequent pipetting. The three most common methods are ethanol precipitation, phenol-chloroform extraction, and minicolumn purification.

  • Ethanol precipitation: this method relies on the principle that DNA is insoluble in alcohol, and so will precipitate out during centrifugation.

  • Phenol-Chloroform extraction: aqueous solutions of DNA samples are mixed with a 1:1 ratio of phenol:chloroform mixture. Given that phenol is immiscible with water, two phases (one aqueous and one phenol) form. During mixing, phenol will force proteins out of the aqueous layer, thus separating DNA from contaminating protein material. The mixture is separated via centrifugation, and the DNA can now be pipetted from the aqueous layer.

  • Minicolumn Purification: this is the most commonly used method, relying on DNA’s ability to bind to solid phase material (such as silica) given the right conditions (pH and salt level in the buffer) and follows a similar procedure as described above. First, a lysis procedure breaks up cellular structures. Then, a buffer solution is added to the spin column to set the conditions to allow DNA to bind to solid phase material (such as silica). Centrifugation forces the DNA to bind to the material, and the solution to pass through. Then, water is added to remove DNA from the solid phase material.

RNA Purification

RNA molecules are special types of nucleic acids that are predominantly single-stranded. Within the last few years RNA molecules - especially non-coding RNAs - have moved into focus of many research projects. RNA molecules are now used in a wide range of applications like gene expression studies, gene knockdowns, gene silencing processes, transcriptome analysis and many more.

RNA Purification is the process of extracting and purifying RNA molecules from samples. The purification procedure follows the same general guidelines as DNA purification, but require a little more attention to detail, as RNases (enzymes which rapidly degrade RNA) are common throughout cells. Their presence can ruin the final yield produced and they require inhibiting for successful RNA purification.

RNA Purification always involves four basic steps:

  • Disruption of cells by adding guanidine thiocyanate and a reducing agent to the sample

  • Breaking of disulphide bonds and inactivation of contaminant proteins by vigorous shaking or vortexing

  • Separation of RNA by adding phenol and chloroform-isoamyl alcohol and centrifugation

  • Washing precipitate with 75% ethanol to remove impurities

Protein Purification

Protein purification and isolation can be some of the hardest challenges in the field of biotechnology and life sciences. At the beginning you have to decide if you need the protein in a biologically active (native) or inactive state for your studies. Next, to choose the best purification technology you should know if your protein is membrane-bonded or free circulating in the cell. Then you have to determine the best environmental conditions e.g. salt concentration, pH of the buffer used to stabilize the structure of the protein.

Proteins are often tagged e.g. with a His-tag to easily purify the protein and boost the purification yield. However, they can also be purified by their size, charge and solubility, but to name a few.

Picking the right protein purification kit will depend on the protein type (species and location in the organism), your downstream application, and will also influence your cloning strategy, if working with fusion proteins.

His-tag and GST-tag protein fusions are commonly used for heterologous protein isolation from E.coli BL21. However, when expressing a eukaryotic protein in prokaryotic systems, you might experience protein degradation and protein precipitation.In this case, or if you plan on examining post translational protein modifications such as protein phosphorylation, you should consider a eukaryotic expression system. While this results in protein modifications that are closer to those of native proteins, they might give some problems when used in combination with some purification tags.

Protein size is also something to consider. The relative size of your tag of choice compared to the protein might interfere with protein folding. For example, it is not advised to use Glutathione S-transferase (GST) fusion for the isolation of small protein for this reason. An alternative would be to use immunoaffinity chromatography, which does not require tagged proteins and can be used for natural sources.

The buffer used in extraction and purification might also interfere with the protein function or protein assays. High detergent concentration can interfere with protein folding which affects protein activity or protein-protein binding. This can be problematic when determining protein concentration, in particular using the Bradford method.

Here at ZAGENO, you have the unique possibility to compare the products from different suppliers to find the most suitable one for your experiment.