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Semester 1: Biochemical and Molecular Biology Techniques
General approaches to biochemical investigation and microscopy techniques
General approaches to biochemical investigation and microscopy techniques
Overview of Biochemical Investigations
Biochemical investigations focus on understanding the biochemical processes within living organisms. Techniques may include enzyme assays, protein purification, and metabolic profiling. These approaches allow for the characterization of biomolecules and their interactions.
Common Biochemical Techniques
Several key techniques are central to biochemical investigations, including spectrophotometry for measuring absorbance, chromatography for separating compounds, and electrophoresis for analyzing proteins and nucleic acids.
Microscopy Techniques in Biochemistry
Microscopy techniques, such as light microscopy, fluorescence microscopy, and electron microscopy, play a crucial role in the visualization of cellular structures. These techniques help in studying the localization of biomolecules and understanding cellular interactions at a microscopic level.
Fluorescence Microscopy
Fluorescence microscopy utilizes fluorescent markers to visualize specific proteins or structures within cells. This method allows for high-resolution imaging and can reveal dynamic processes in live cells.
Electron Microscopy
Electron microscopy provides high-resolution imaging of cells and tissues. Transmission electron microscopy (TEM) allows for detailed internal structure visualization, while scanning electron microscopy (SEM) offers surface topography insights.
Integration of Biochemical Techniques with Microscopy
Integrating biochemical techniques with microscopy enhances our understanding of cellular processes. Techniques such as immunofluorescence combine the sensitivity of biochemical assays with the visual power of microscopy.
Applications in Research and Medicine
Biochemical investigations and microscopy techniques have vast applications in research, diagnostics, and therapeutics. They are essential in areas such as drug development, disease diagnosis, and understanding cellular mechanisms.
Chromatographic techniques: adsorption, partition, affinity chromatography, HPLC
Chromatographic techniques
Adsorption Chromatography
Adsorption chromatography is based on the principle of adsorption where the separation of components occurs due to their differential adsorption on a stationary phase. Common stationary phases include silica gel or alumina. The sample is applied onto the stationary phase, and the mobile phase, which can be a liquid or gas, carries the sample through. Components that have a higher affinity for the stationary phase move more slowly, allowing for separation.
Partition Chromatography
Partition chromatography relies on the partitioning of compounds between two liquid phases: a stationary phase supported on a solid (often called a support) and a moving solvent. The separation occurs because of the different solubilities of analytes in the two phases. This technique is widely used for separating amino acids, sugars, and other small molecules.
Affinity Chromatography
Affinity chromatography is a technique that involves the specific interaction between a target molecule and a ligand immobilized on a stationary phase. This selectivity allows for the purification of proteins, nucleic acids, and other biomolecules. The elution process commonly uses a solution that competes with the target molecule for binding to the ligand, leading to high purity and concentration of the target.
High-Performance Liquid Chromatography (HPLC)
HPLC is a highly efficient technique for separating, identifying, and quantifying compounds in a mixture. It operates under high pressure, allowing for smaller particle sizes of the stationary phase and leading to better resolution. HPLC is versatile, applicable in pharmaceuticals, environmental testing, and biochemical analysis. Different types of HPLC include reversed-phase, normal-phase, and ion-exchange chromatography.
Electrophoretic techniques: PAGE, SDS-PAGE, isoelectric focusing, capillary electrophoresis
Electrophoretic techniques
PAGE
PAGE stands for Polyacrylamide Gel Electrophoresis. This technique separates proteins based on their size and charge. In PAGE, a polyacrylamide gel provides a medium for the movement of molecules. Smaller molecules migrate faster through the gel matrix than larger ones. PAGE is widely used for the analysis and purification of proteins and nucleic acids.
SDS-PAGE
SDS-PAGE refers to Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. This method incorporates SDS, an anionic detergent, which denatures proteins, giving them a uniform negative charge proportional to their mass. SDS-PAGE is particularly useful for estimating protein molecular weights and is a standard technique in protein biochemistry.
Isoelectric focusing
Isoelectric focusing is a technique that separates proteins based on their isoelectric points (pI). In this process, a pH gradient is established in a gel, and proteins migrate until they reach a point where their net charge is zero. This technique is highly effective for resolving proteins with similar sizes but different charges.
Capillary electrophoresis
Capillary electrophoresis is a high-resolution technique that separates ions based on their charge-to-size ratio using a thin capillary tube filled with an electrolyte. It offers rapid analysis and high sensitivity. Due to its efficiency, it is increasingly used in various applications, including drug testing and genomics.
Spectroscopic techniques: UV-Visible, IR, ESR, NMR, Mass spectrometry, atomic absorption
Spectroscopic techniques
UV-Visible Spectroscopy
Technique used to measure the absorption of ultraviolet or visible light by a substance. It is useful for determining the concentration of analytes in solution and helps in studying electronic transitions in molecules.
Infrared (IR) Spectroscopy
Technique that measures the absorption of infrared radiation by a sample. It provides information about molecular vibrations and functional groups in organic compounds, making it valuable for qualitative and quantitative analysis.
Electron Spin Resonance (ESR) Spectroscopy
Technique used to detect unpaired electrons in a sample. It provides information about the electronic structure of radicals and paramagnetic species, playing a crucial role in studying reaction mechanisms.
Nuclear Magnetic Resonance (NMR) Spectroscopy
Technique that exploits the magnetic properties of certain nuclei. It is used to determine the structure of organic compounds, study molecular dynamics, and obtain information about molecular interactions.
Mass Spectrometry
Technique used to determine the mass-to-charge ratio of ions. It provides information about molecular weight, structure, and composition, and is widely used in proteomics, metabolomics, and organic chemistry.
Atomic Absorption Spectroscopy (AAS)
Technique used to analyze the concentration of metal ions in a sample by measuring the absorption of light. It is pivotal in environmental testing, food analysis, and clinical diagnostics.
Radiolabeling and centrifugation techniques: principles and applications
Radiolabeling and centrifugation techniques: principles and applications
Principles of Radiolabeling
Radiolabeling involves attaching a radioactive isotope to a molecule, which can be tracked or measured. Common isotopes used include Carbon-14, Tritium, and Iodine-125. The choice of isotope depends on the specific application, such as the type of study or the detection method.
Methods of Radiolabeling
Radiolabeling can be achieved through various methods, including direct labeling, where isotopes are incorporated directly into the molecule, and indirect labeling, where a chelator is used to bind the radioactive isotope without modifying the molecule significantly.
Applications of Radiolabeling
Radiolabeling is widely used in fields such as biochemistry and molecular biology for tracing metabolic pathways, studying receptor interactions, and imaging in medical diagnostics. It provides valuable insights into the dynamics of biological processes.
Principles of Centrifugation
Centrifugation is a technique that employs centrifugal force to separate components of different densities in a mixture. The principles are based on sedimentation, where particles are forced to move outward from the axis of rotation.
Types of Centrifugation
There are several types of centrifugation, including differential centrifugation, which separates components based on size and density, and density gradient centrifugation, which allows for the separation of particles based on buoyancy in a gradient medium.
Applications of Centrifugation
Centrifugation is used in various applications such as isolating cellular organelles, purifying proteins, and separating blood components. It is a fundamental technique in laboratories for the preparation of samples.
Integration of Radiolabeling and Centrifugation
Radiolabeling can be combined with centrifugation techniques for enhanced analysis of labeled molecules. For instance, after radiolabeled compounds are introduced into a biological sample, centrifugation can help isolate and study their distribution and activity.
