Opinion Article - Journal of Clinical and Bioanalytical Chemistry (2021) Volume 5, Issue 5
Gel Electrophoresis usage in separation of proteins
Associate professor, Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy Cluj- Napoca, Romania
- *Corresponding Author:
- Hanganu Daniela
Associate professor Department of Pharmacognosy Faculty of Pharmacy University of Medicine and Pharmacy Cluj-Napoca Romania
E-mail: [email protected]
Accepted Date: December 16, 2021;
Citation: Daniela H. Gel electrophoresis usage in separation of proteins. J Clin Bioanal Chem. 2021;5(5): 07.
Electrophoresis is the movement of dispersed particles with respect to a liquid under the influence of a spatially uniform electric field. Electrophoresis is used in the laboratory to separate macromolecules by size. This technique applies a negative charge so that the protein moves towards a positive charge. Electrophoresis is widely used in the analysis of DNA, RNA, and proteins. Gel electrophoresis is a technique for separating and analysing macromolecules (DNA, RNA, and proteins) and their fragments based on size and charge. It separates by charge or size (IEF agarose, essentially size independent) in clinical chemistry and by length in a mixed population of DNA and RNA fragments in biochemistry and molecular biology. Used to estimate the size of. Or the protein is separated by electric charge. Nucleic acid molecules are separated by the application of an electric field in order to move negatively charged molecules through a matrix of agarose or other substances. Short molecules move faster and more than long molecules because short molecules move more easily through the pores of the gel. This phenomenon is called 7. The protein is separated by the load of agarose because the pores of the gel are too small to sift the protein. Nanoparticles can also be separated using gel electrophoresis. Gel electrophoresis uses gel as an anti-convection or sieving medium during electrophoresis. This is the movement of charged particles in an electric current. The gel also acts as a sieving medium that suppresses the thermal convection caused by the application of an electric field and delays the passage of molecules. Gels can also be used to maintain final separation so that staining can be applied after electrophoresis. DNA gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA by polymerase chain reaction (PCR), but mass analysis, RFLP, PCR, cloning, DNA sequencing for further characterization or Southern blotting.
Types of gel
1) The most common types of gels used are agarose and polyacrylamide gels. Each type of gel is suitable for different types and sizes of analytes. Polyacrylamide gels are commonly used for proteins and have very high resolution for small DNA fragments (5500 bp). Agarose gels, on the other hand, are commonly used for DNA fragments sized at 50-20,000 bp due to their lower DNA resolution but wider separation range, but pulsed-field gel electrophoresis has a resolution greater than 6 Mb. It is possible (PFGE). Polyacrylamide gels operate in a vertical configuration, while agarose gels normally operate horizontally in underwater mode. Also, while agarose is thermoset, polyacrylamide is produced by a chemical polymerization reaction, so the casting method is also different.
After the electrophoresis is complete, the molecules in the gel can be stained and visualized. DNA can be visualized using ethidium bromide. Ethidium bromide fluoresces under UV light when inserted into DNA, but proteins can be visualized by silver staining or Coomassie brilliant blue dye. Other methods can be used to visualize the separation of the components of the mixture on the gel. If the molecule to be separated contains radioactivity, for example a DNA sequencing gel, you can record an autoradiogram of the gel. In many cases, you can use the Gel Doc system to take a picture of the gel.
After separation, additional separation methods such as isoelectric focusing and SDS PAGE can be used. The gel is then physically cleaved to extract the protein complex from each serving separately. Each extract can then be analyzed, for example, by peptide mass fingerprinting or de novo peptide sequencing after in-gel digestion. It can provide a lot of information about the identity of the proteins in the complex.
• Estimating the size of DNA molecules after restriction enzyme digestion can be done. Restricted mapping of cloned DNA.
• Analysis of PCR products such as molecular genetic diagnosis and genetic fingerprints can be performed.
• Separation of restricted genomic DNA/RNA before Southern blotting can be performed.
• Gel electrophoresis is used in forensic medicine, molecular biology, genetics, microbiology, and biochemistry. By visualizing the gel with UV and gel imaging devices, the results can be analyzed quantitatively. The image is taken with a computer controlled camera, the intensity of the band or spot of interest is measured and compared to a standard or marker loaded on the same gel. Measurements and analyzes are usually performed using special software.