Protein Separation Techniques and G6PD Overview

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Questions and Answers

Why are protein isolation techniques important in analysis of proteins? Give TWO (2) reasons.

Protein isolation techniques are important for isolating and purifying proteins of interest, separating them from complex mixtures, and enabling detailed analysis and characterization of their structure, function, and interactions.

List GENERAL steps in protein purification.

The general steps in protein purification include: 1. Cell lysis or disruption to release proteins. 2. Removal of debris and nucleic acids. 3. Initial fractionation (e.g., salting out, precipitation). 4. Affinity chromatography. 5. Size exclusion chromatography. 6. Ion exchange chromatography. 7. Concentration and buffer exchange.

Briefly explain on various sources of sample for protein purification.

Various sources of samples for protein purification include: 1. Cell cultures (e.g., bacteria, yeast, mammalian cells) for recombinant protein production. 2. Tissues and organs from organisms. 3. Biological fluids (e.g., blood, serum, plasma, milk). 4. Plant extracts.

Briefly explain on disruption method of samples/specimen in protein purification.

Disruption methods for samples/specimens in protein purification involve techniques like sonication, mechanical homogenization, enzymatic lysis, and chemical lysis to break open cells or tissues, releasing proteins into a solution for further processing. Signup and view all the answers

List TWO (2) techniques for separating bulk mixtures in protein purification.

Two techniques for separating bulk mixtures in protein purification are: 1. Salting out (ammonium sulfate precipitation). 2. Centrifugation. Signup and view all the answers

How can you determine the structure of a complex molecule from a single crystal via X-rays crystallography technique?

The structure of a complex molecule can be determined from a single crystal via X-ray crystallography technique by diffracting X-rays through the crystal, analyzing the resulting diffraction pattern, and using mathematical algorithms to calculate the electron density map, which reveals the arrangement of atoms in the molecule. Signup and view all the answers

Briefly explain about the preparation for producing protein crystals.

The preparation for producing protein crystals involves: 1. Purifying the protein to high homogeneity. 2. Concentrating the protein solution. 3. Screening various crystallization conditions (e.g., pH, salt concentration, precipitant) to find suitable conditions that promote crystal formation. 4. Growing crystals using techniques such as vapor diffusion or microbatch crystallization. Signup and view all the answers

Briefly explain about the key processes involved in the crystallisation laboratory.

The key processes involved in the crystallization laboratory include: 1. Protein purification and preparation. 2. Crystallization screening and optimization. 3. Crystal harvesting and cryoprotection. 4. X-ray diffraction data collection. 5. Structure determination and refinement. Signup and view all the answers

Give FOUR (4) examples of the molecules used in this video for 3D structures generation via X-rays crystallography technique.

Four examples of molecules used for 3D structure generation via X-ray crystallography technique include proteins, enzymes, DNA fragments, and small organic molecules. Signup and view all the answers

Briefly explain about the use of X-rays radiation in predicting the structure of molecules.

X-ray radiation is used to predict the structure of molecules by diffracting through a crystal of the molecule, producing a diffraction pattern. This pattern is analyzed to determine the arrangement of atoms within the molecule, allowing scientists to construct a 3D model of its structure. Signup and view all the answers

What were the limitations in earlier technique invented by the crystallographer scientist, William Henry Bragg (1920s) for X-rays generator?

The limitations in the earlier technique invented by William Henry Bragg (1920s) for X-ray generators included low intensity of X-ray beam and limited resolution, which hindered the analysis of complex molecular structures. Signup and view all the answers

What are the advancements made nowadays by using synchrotrons for X-rays generator.

Advancements made nowadays by using synchrotrons for X-ray generators include: 1. Higher intensity X-ray beams, leading to better data quality. 2. Tunable X-ray wavelengths, allowing for anomalous diffraction experiments. 3. Faster data collection times. 4. Improved resolution for studying larger and more complex structures. Signup and view all the answers

Briefly explain the whole event starts from the core of synchrotrons until producing X-rays radiation and later to perform diffraction patterns analysis.

The process starts with electrons being accelerated to near the speed of light in a synchrotron storage ring. These electrons are then forced to change direction by powerful magnets, causing them to emit intense beams of X-rays. The X-rays are directed onto a crystal of the molecule being studied, and the resulting diffraction pattern is captured by a detector. Finally, the diffraction pattern is analyzed using computational methods to determine the 3D structure of the molecule. Signup and view all the answers

Briefly explain the association between diffraction patterns analysed with the predicted 3D structure of molecules.

The diffraction patterns are mathematically analyzed to determine the position and intensity of the diffracted X-rays, which are then used to calculate the electron density map of the molecule. This map reveals the arrangement of atoms in the molecule, allowing scientists to build a 3D model of its structure. Signup and view all the answers

Briefly explain about the biological function of glucose-6-phosphate dehydrogenase (G6PD).

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that plays a crucial role in the pentose phosphate pathway, which produces NADPH. NADPH is essential for protecting cells from oxidative damage by reducing oxidative stress and maintaining the supply of reduced glutathione. Signup and view all the answers

What is selective advantage of G6PD deficiency?

The selective advantage of G6PD deficiency is that it provides some protection against malaria. Individuals with G6PD deficiency are less susceptible to malarial infection because the lower levels of NADPH in their red blood cells make them less hospitable for the malaria parasite to grow and reproduce. Signup and view all the answers

Briefly explain about severity level of G6PD.

The severity level of G6PD deficiency varies depending on the specific genetic mutation. Some individuals with G6PD deficiency may be asymptomatic or have mild symptoms, while others may experience severe hemolytic anemia, especially when exposed to oxidative stressors. Signup and view all the answers

Briefly explain on G6PD deficiency presentation in neonatal jaundice.

G6PD deficiency can present in neonatal jaundice as increased bilirubin levels due to hemolysis of red blood cells. This can lead to jaundice, which, if severe, may cause brain damage (kernicterus) if not promptly treated. Signup and view all the answers

Briefly explain about fluorescent spot test in blood test.

The fluorescent spot test is a screening test for G6PD deficiency in blood. It measures the production of NADPH by G6PD. A blood sample is applied to a filter paper, and after incubation and exposure to UV light, the presence or absence of fluorescence indicates the level of G6PD activity. Signup and view all the answers

Briefly explain about the typical clinical condition called haemolytic anaemia observed in patient with G6PD deficiency.

Hemolytic anemia in G6PD deficiency occurs when red blood cells are prematurely destroyed due to oxidative stress. This happens because the lack of G6PD impairs the cells' ability to protect themselves from reactive oxygen species, leading to cell damage and rupture. Signup and view all the answers

Name the foods that are safe to be consumed by patient with G6PD deficiency.

Foods generally considered safe for individuals with G6PD deficiency include most non-legume vegetables, fruits, grains, lean meats, and dairy products. Signup and view all the answers

How you relate oxidative stress in erythrocytes with G6PD function?

Oxidative stress in erythrocytes is related to G6PD function because G6PD is essential for producing NADPH, which protects red blood cells from oxidative damage. When G6PD is deficient, red blood cells are more vulnerable to oxidative stress, leading to hemolysis. Signup and view all the answers

Briefly explain in diet management programmes, why certain foods are recommended to be avoided by patient with G6PD deficiency.

In diet management programs, certain foods are recommended to be avoided by patients with G6PD deficiency because they contain compounds that can induce oxidative stress or trigger hemolytic episodes. Avoiding these foods helps reduce the risk of red blood cell damage and anemia. Signup and view all the answers

Give FOUR (4) your recommendations to patients with G6PD deficiency for their healthy lifestyles.

Four recommendations for patients with G6PD deficiency for their healthy lifestyles include: 1. Avoid known triggers such as fava beans and certain medications. 2. Stay hydrated. 3. Maintain a balanced diet rich in antioxidants. 4. Consult healthcare professionals before taking any new medications or supplements. Signup and view all the answers

Flashcards

Protein Purification Importance

Protein isolation techniques are crucial for analyzing proteins to understand their structure and function.

General Steps in Protein Purification

The common steps include sample collection, extraction, precipitation, and purification.