Lecture 8 - Serum Proteins and diagnosis Flashcards
Why is serum protein separation used in clinical practice?
Serum protein separation is used in clinical practice for various purposes, including identifying diseases causing overproduction or underproduction of specific proteins and pinpointing affected body parts through specific isoenzyme identification.
List some methods used for separating serum proteins in clinical practice.
Methods for separating serum proteins include cellulose acetate electrophoresis, SDS-PAGE, native PAGE, and newer technologies like isoelectric focusing, 2D page, and capillary zone electrophoresis.
What is electrophoresis, and how does it work?
Electrophoresis is the movement of charged molecules or ions in an electric field. Positively charged ions move towards the cathode, and negatively charged ions move towards the anode. Different components in a mixture, varying in size, mass, and charge, separate based on their migration through a medium.
Explain cellulose acetate electrophoresis and why it’s used.
Cellulose acetate electrophoresis is conducted at a specific pH to give all proteins a negative charge, causing them to move towards the anode. This method is quick, cost-effective, requires only a small sample quantity, and provides rapid staining.
Describe polyacrylamide gel electrophoresis (PAGE) and its principle.
Polyacrylamide gel electrophoresis involves creating a polyacrylamide gel with different pore sizes by varying the gel’s concentration. This allows the separation of proteins based on their sizes.
Explain SDS-PAGE and its role in separating proteins.
SDS-PAGE involves incubating native proteins with mercaptoethanol and SDS to disrupt S-S bridges and give proteins a negative charge. This process results in linear structures with negative shells that migrate through the gel, separating proteins based on their size.
What stains can be used to visualize proteins in electrophoresis gels?
Various stains can be used, including Coomassie blue (hydrophobic and negatively charged), fluorescent stains like sypro orange and red, and silver staining at very low protein concentrations.
Explain activity staining and provide an example.
Activity staining, such as zymography, involves using enzymatic activity to identify a protein. For instance, lactate dehydrogenase (LDH) activity can be visualized by converting NBT (nitroblue tetrazolium) into insoluble form in the gel.
What is isoelectric focusing (IEF), and how does it separate proteins?
IEF separates proteins based on their isoelectric point (pI), which is the pH at which there is no net charge on the protein. Proteins migrate to the part of the gel where pH equals their pI, and they stop moving because of no net charge. IEF offers high-resolution protein separation and is useful in diagnosing conditions like multiple sclerosis.
Explain 2D PAGE and its utility.
2D PAGE combines isoelectric focusing (separation based on pI) with SDS-PAGE (separation based on size). It is useful for comparing healthy and diseased states. Computer software is required for analysis, and proteins can be identified by cutting out spots, digesting them, and performing identification.
What is capillary electrophoresis, and how does it work in analyzing serum proteins?
Capillary electrophoresis, specifically capillary zone electrophoresis (CZE), uses a narrow bore capillary with silica on the inside and a polymer on the outside to analyze serum proteins. All proteins move towards the cathode, and their movement is detected by a computer. CZE offers high speed, high resolution, and uses small sample volumes.
Describe the electrophoretic patterns of serum proteins and their significance.
Serum proteins’ electrophoretic patterns reveal distinct peaks, including albumin, α1, α2, β, and γ. These patterns can change in response to various medical conditions, helping with diagnosis and monitoring.
Discuss the role of albumin and its significance in diagnosing various conditions.
Albumin, synthesized in the liver, maintains plasma oncotic pressure and serves as a transport protein for substances like calcium and bilirubin. Low albumin levels can lead to oedema and may indicate liver disease, nephrotic syndrome, or malnutrition.
Explain how immunoglobulins are analyzed and how their presence or absence can be indicative of specific diseases.
Immunoglobulins (Ig) can be analyzed by treating serum with antisera to different Ig types. Specific diseases can be indicated by the presence or absence of particular Ig types. For example, multiple myeloma can be diagnosed by the presence of monoclonal Ig, its quantity, and type, with Bence Jones proteins present in the urine.
