Agarose gel electrophoresis is one of the most widely used techniques in molecular biology laboratories. Through electrophoresis we can separate DNA and RNA fragments based on their size and charge, visualize them by means of a simple stain, and thus determine the nucleic acid content of a sample.
How is gel electrophoresis carried out?
The porous agarose gel is used to separate macromolecules of various sizes. The gel is immersed in a buffer solution in an electrophoresis chamber. The chamber has two electrodes – one positive and one negative – at its two ends.
Samples are loaded into tiny wells on the gel with the help of a pipette. Once the samples have been fully loaded, electrical current is applied. Now, the charged molecules present in the sample begin to migrate through the gel towards the electrodes. Negatively charged molecules move towards the positive electrode and positively charged molecules migrate towards the negative electrode.
The speed at which each molecule travels through the gel is called its electrophoretic mobility and is mainly determined by its net charge and size. Strongly charged molecules move faster than weakly charged ones. Smaller molecules run faster, leaving behind larger ones. Thus, strong charge and small size increase the electrophoretic mobility of a molecule, while weak charge and large size decrease the mobility of a molecule.
When the separation is complete, the gel is stained with a dye to reveal the separation bands. Ethidium bromide is a fluorescent dye commonly used in gel electrophoresis. The gel is soaked in a dilute ethidium bromide solution and then placed in a UV transilluminator to visualize the separation bands. Bands are immediately inspected or photographed for future reference.
What is protein purification?
Protein purification is a laboratory technique that consists of a series of steps, which aim to isolate a single type of protein from a complex mixture. This technique is essential for the characterization of function, structure, and interactions.
The starting material is usually a biological tissue or microbial culture. This purification is carried out in a series of stages that can be summarized:
- Extract the protein from the matrix that confines it
- Separate the protein and non-protein parts of the mixture
- Separate the desired protein from other proteins
Steps for the purification of a protein
- Create a crude protein extract: Crude intracellular protein extracts are prepared by lysing cells, using chemical or mechanical processes. Extracellular proteins are obtained by centrifuging the solution and removing the cells.
- Intermediate purification: This can be done by precipitating proteins with a highly concentrated salt such as ammonium sulfate. The protein salts are then passed through a semi-permeable membrane in a step known as dialysis or subjected to chromatography or gel exclusion filtration.
- Final purification: This is achieved through affinity chromatography, polyacrylamide gel electrophoresis or immunoblotting.
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