Proteins secreted by bacteria can be used as biomarkers for diagnostics. Give a concise account of how you can use a 2D gel electrophoresis combined with mass spectrometry to develop a diagnostic for E. coli infection. Discuss how you could extend this technique to develop a diagnostic for COVID-19 based on protein electrophoresis?
Proteins secreted by bacteria can be used as biomarkers for diagnostics. Give a concise account of how you can use a 2D gel electrophoresis combined with mass spectrometry to develop a diagnostic for E. coli infection. Discuss how you could extend this technique to develop a diagnostic for COVID-19 based on protein electrophoresis?
The identification of protein antigens from pathogens can serve as a diagnostic tool. Several pathogen-specific antigens are detected by biophysical techniques. However, simpler techniques like SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) cannot detect proteins with a minor difference in their molecular weight. The more complex version of this technique called the 2D (dimensional) gel electrophoresis can be utilized to detect and separate proteins having similar molecular weight.
- In 2D gel electrophoresis, the protein mixture from the lysate of bacterial culture is separated on the basis of their isoelectric point and molecular weight. The appropriate clinical sample like blood, urine, sputum, or stool can be taken. This sample should be processed to isolate the pathogenic disease-causing microbe by using bacterial culture techniques. The bacterial culture can then be lysed to release the proteins that are later denatured with urea.
- In the first dimension, the bacterial cell extract is run on a polyacrylamide gel. This gel contains a pH (potential of hydrogen) gradient. The proteins in this gel migrate under the electric field and attain a zero charge whenever they reach a region having pH equal to their pI (isoelectric point). Therefore, the proteins in the extract stop migrating whenever they reach their respective pI values.
- Next, this gel is placed horizontally on a second polyacrylamide gel that is saturated with SDS (sodium dodecyl sulfate). SDS will impart a negative charge on all the protein to ensure that they separate only according to their molecular weight. The resolution of proteins based on their charge and molecular weight ensures efficient protein separation.
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