3 Protein Analysis Techniques Explained
Proteins, also known as polypeptides, are organic compounds made up of amino acids. They are arranged in a linear chain and folded into a globular form. Proteins are essential parts of organisms and they participate in virtually every process within cells. Many proteins are enzymes that catalyse biochemical reactions and are vital to metabolism. The size of a protein is an important physical characteristic and scientists often use particle size analysers in their studies to discuss protein size or molecular weight.
The Structure of Proteins
Most proteins fold into unique three-dimensional structures. The shape that a protein folds into naturally is known as its native conformation. While most proteins can fold unassisted through the chemical properties of their amino acids, others require the aid of molecular chaperones. There are four distinct aspects of a protein’s structure:
- Primary structure: the amino acid sequence
- Secondary structure: regularly repeating local structures stabilised by hydrogen bonds
- Tertiary structure: the overall shape of a single protein molecule; the spatial relationship of the secondary structures to one another
- Quaternary structure: the structure formed by several protein molecules which function as a single protein complex
Protein Analysis Techniques
Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. Hence, they have different molecular structures, nutritional attributes and physiochemical properties. There are three major protein analysis techniques, protein separation, western blotting and protein identification.
1. Protein Separation
- SDS-PAGE: This method separates proteins mainly on the basis of molecular weight as opposed to charge or folding. It is a technique that is widely used in biochemistry, forensics, genetics and molecular biology.
- Isoelectric Focussing: In this method, different molecules are separated by their electric charge differences. This technique is a type of zone electrophoresis that is usually performed in a gel and takes advantage of the fact that a molecule’s charge changes with the pH of its surroundings.
- Chromatic Methods: There are two chromatic methods frequently used for protein separation – high-performance liquid chromatography and thin-layer chromatography. Both these methods are particularly useful adjuncts to gel-based approaches. Although chromatography is a common technique in biochemistry laboratories used for purification, identification and quantification of protein mixtures, laser diffraction is traditionally used for pre-column size and polydispersity management.
- Two-dimensional Gel Electrophoresis: This is a powerful gel-based method commonly used to analyse complex samples in the interest of characterising the full range of proteins in the sample, not just a few specific proteins.
2. Western Blotting
The most common version of this method is immunoblotting. This technique is used to detect specific proteins in a given sample of tissue homogenate or extract. The sample of proteins is first electrophoresed by SDS-PAGE to separate the proteins based on molecular weight. The proteins are then transferred to a membrane where they are probed using antibodies specific to the target protein.
3. Protein Identification
There are two methods that are commonly used to identify proteins.
- Edman Degradation: Developed by Pehr Edman, this is a method of sequencing amino acids in a peptide. Here, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues.
- Mass Spectrometry: An analytical technique that measures the mass-to-charge ratio of charged particles, mass spectrometry is used for determining masses of particles and the elemental composition of a sample of molecule as well as for elucidating the chemical structure of molecules such as peptides.
Protein analysis made easy
Choosing the best protein analysis technique and the equipment to use to carry it out can be difficult. ATA Scientific can assist you with finding the best equipment, so contact us today for a free consultation.