Protein is a primary component of tissues and cells. And when one analyzes a tissue for research, protein estimation is inevitable.
Protein analysis helps
- Quantification of tissue or cell material in biological research.
- Understand body physiology
- For diagnosis of diseases by western blot, ELISA.
- To determine the mechanism of disease.
- Quality control of packed food material and protein supplements.
- To develop vaccines.
- Biosynthesis of Hormones and Enzymes
- Forensic Investigation
- Identification of bacteria.
Protein is a widely distributed biomolecule in the body.
These proteins exist in different forms like amino acids, peptides, polypeptides, enzymes, hormones, etc.
They carry out various functions in the body, and almost all body reactions occur due to the involvement of protein.
Protein structure can be a single chain molecule, a 3-dimensional structure, or even a bundle or complex form.
Why is protein analysis important
For Quantification of tissue or cell material
- In biological research, cell contents and tissues are analyzed many times to study their physiology, morphology, or other changes.
- This analysis is finally quantified in terms of per gram of protein or per microgram of protein.
- This analysis of protein from the tissue sample is done by using the folin reagent method of Lowry.
- Previously, the measurements were expressed as a gram of tissue or biological sample.
For diagnosis of diseases
- Proteins are the major biochemicals that are widely used for the diagnosis of diseases.
- They can be estimated from tissue and blood samples of a diseased patient.
- For example, diseases like HIV, hepatitis, and more can be estimated by ELISA and western blot techniques.
- Besides, hemoglobin analysis in the blood is done to check for anemia.
- The normal range for an adult woman is 12 to 16 gm/dL; for men, it is 13 to 17 gm/dL. Below those levels is termed anemia.
To Understand Body Physiology
- Since proteins are everywhere in the body, their analysis helps us understand the body’s function.
- For example, hemoglobin is a protein in the red blood cell with iron in it.
- This iron helps to bind oxygen molecules.
- The change in the structural configuration of this protein, along with iron, is the reason for gaseous exchange.
- Similarly, the digestion of proteins in the digestive tract is done by the breakdown of proteins into peptides and amino acids.
To determine the mechanism of disease.
- Many diseases have changes in protein physiology or nature.
- By analyzing proteins in the tissue, the disease mechanism can be understood.
- This helps to tackle the disease better with targeted drugs.
- For example, in Alzheimer’s disease, beta-amyloid protein plaques are formed. These proteins are toxic and disrupt nerve function.
Quality control of packed food and protein supplements.
- Analysis of protein content is mandatory for the packed food industry.
- You can notice the labels of packed foods containing protein, carbohydrates, and fats.
- This is necessary to inform the consumer about what the packet contains.
- Similarly, in protein supplements, the content of protein is indicated on the box.
- Many seek a product with more than 99% of protein.
To develop vaccines
- Vaccines are the immune-modifying substance that is used to develop disease resistance.
- These vaccines contain a protein structure called an epitome, which can trigger an immune response in the host body.
- This leads to the formation of antibodies against that protein and can act as a defense for future exposure to that antigen from pathogens.
- So, to develop a suitable antigen that can trigger the human immune system, one needs to study the protein structure of the pathogen.
Biosynthesis of Hormones and Enzymes
- Diseases like diabetes and anemia require hormones like Insulin and erythropoietin for treatment.
- Previously, insulin was obtained from animals. A clear study of the molecular structure reveals a difference in the amino acid of human and bovine insulin.
- So, the exact human hormones are prepared by rDNA technology using fermentation.
- During preparation, chains of peptides are synthesized and then brought into a structural confirmation as the original hormones.
- This protein synthesis can be done by analyzing the protein structure before the actual method is designed for the entire process.
Forensic Investigation
- When there is a crime scene, most of the clues are collected, including the hair, blood, other body tissue, and fluid samples.
- These samples have protein in them and are analyzed to investigate the crime.
Identification of bacteria
- Bacterial identification is mostly made by cultures.
- But, this is a tedious and time-consuming process.
- Protein analysis using mass spectrometry is a faster and easier process.
Protein analysis & quantification
- Protein, being a biological entity, is analyzed to determine the quantity and quality of protein and, thereby, the state of biological reaction or process.
They can be analyzed by methods like
- Folin reagent by Lowry.
- Chemical methods of identification.
- Spectroscopic methods.
and the approach is different based on the need, like
- Protein sequence analysis
- Protein structure analysis
- Protein binding analysis
- Crude protein analysis (quantification of protein).
Protein sequence analysis
- This is necessary to analyze the sequence of amino acids or peptides in proteins like Insulin.
- Insulin was previously used from animals like pigs and cattle for diabetes treatment.
- However, this insulin had side effects due to the difference in the type of amino acids in the protein sequence.
- Hence, biotechnology solved the problem by providing insulin with an exact sequence that is identical to human insulin with no side effects.
Protein structure analysis:
- Proteins have different structures, such as secondary, tertiary, Quaternary, and 3-fold structures.
- These structures influence the protein’s function; for example, 3-dimensional ones can carry some substances by binding them over the surface, encasing them inside their structure, etc.
- Chain and simple structure proteins easily pass through cell walls.
- This can be done by mass spectrometry, NMR spectroscopy, and X-ray crystallography.
Crude protein analysis
- This is done qualitatively, i.e., to know the nature and type of protein and impurities.
- A qualitative test for protein indicates the type of protein and is done by chemical methods.
- It is also done quantitatively, whereby the amount of protein in a given biological sample is known.
- However, the Lowry method is widely followed for the quantification of protein.
- This is a colorimetric procedure wherein the protein sample is treated to produce a dense color as a protein function.
- This color intensity is then estimated using spectrophotometry to determine the quantity of protein.
Techniques involved:
Most protein analyses use calorimetry and, to some extent, chromatography, electrophoresis, Western blot, and Elisa techniques.
Colorimetry
- It involves the development of color and estimation using a spectrophotometer.
- This is a relatively fast, simple, and inexpensive method.
Chromatography
- The protein samples are separated and then estimated using spectrophotometric methods in chromatography.
- This technique is preferred when the same set of proteins is present or when a protein is hard to isolate from the given sample.
- Methods like thin-layer chromatography can be used to estimate rough protein.
Electrophoresis
- This is a highly specialized method where proteins of different molecular weights are separated under the influence of electricity.
- Then, the separated bands are analyzed using immune-labeled enzymes or spectrophotometry.
Western blot
- This method also relies on separating proteins from a sample.
- They are then analyzed using immune blotting or spectrometry methods.
Elisa
This is a technique that helps identify one specific protein of interest.
It relies on an antibody-antigen reaction and can be done fast.