advanced higher bio

• Health and safety


  • Substances, organisms, and equipment in a laboratory can present a hazard
  • Hazard, risk, and control of risk in the lab by risk assessment
• Liquids and solutions


  • Method and uses of linear and log dilution
  • Production of a standard curve to determine an unknown
  • Use of buffers to control pH
  • Method and uses of a colorimeter to quantify concentration and turbidity
• Separation techniques


  • Use of centrifuge to separate substances of differing density
  • Paper and thin layer chromatography can be used for separating different substances such as amino acids and sugars
  • Principle of affinity chromatography and its use in separating proteins
  • Principle of gel electrophoresis and its use in separating proteins and nucleic acids
  • Native gels separate proteins by their shape, size and charge
  • SDS–PAGE separates proteins by size alone
  • Proteins can be separated from a mixture using their isoelectric points (IEPs)
  • Proteins can also be separated using their IEPs in electrophoresis
• Detecting proteins using antibodies

  • Immunoassay techniques are used to detect and identify specific proteins
  • These techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies
  • An antibody specific to the protein antigen is linked to a chemical 'label'
  • Western blotting is a technique, used after SDS–PAGE electrophoresis
  • The separated proteins from the gel are transferred (blotted) onto a solid medium
  • The proteins can be identified using specific antibodies that have reporter enzymes attached
• Microscopy


  • Bright-field microscopy is commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
  • Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
• Aseptic technique and cell culture

  • Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells
  • A microbial culture can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients
  • Animal cells are grown in medium containing growth factors from serum
  • In culture, primary cell lines can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions
  • Plating out of a liquid microbial culture on solid media allows the number of colony-forming units to be counted and the density of cells in the culture estimated
  • Serial dilution is often needed to achieve a suitable colony count
  • Method and use of haemocytometer to estimate cell numbers in a liquid culture
  • Vital staining is required to identify and count viable cells
• The proteome


  • The proteome is the entire set of proteins expressed by a genome
  • The proteome is larger than the number of genes, particularly in eukaryotes, because more than one protein can be produced from a single gene as a result of alternative RNA splicing
  • Not all genes are expressed as proteins in a particular cell type
  • The set of proteins expressed by a given cell type can vary over time and under different conditions
• Synthesis and transport of proteins

  1. Intracellular membranes

  2. Synthesis of membrane components

  3. Movement of proteins between membranes

  4. The secretory pathway
• Intracellular membranes


  • Eukaryotic cells have a system of internal membranes, which increases the total area of membrane
  • The endoplasmic reticulum (ER) forms a network of membrane tubules continuous with the nuclear membrane
  • The Golgi apparatus is a series of flattened membrane discs
  • Lysosomes are membrane-bound organelles containing a variety of hydrolases that digest proteins, lipids, nucleic acids and carbohydrates
  • Vesicles transport materials between membrane compartments
• Synthesis of membrane components

  • Lipids and proteins are synthesised in the ER
  • Lipids are synthesised in the smooth endoplasmic reticulum (SER) and inserted into its membrane
  • The synthesis of all proteins begins in cytosolic ribosomes
  • The synthesis of cytosolic proteins is completed there, and these proteins remain in the cytosol
  • Transmembrane proteins carry a signal sequence, which halts translation and directs the ribosome synthesising the protein to dock with the ER, forming RER
  • Translation continues after docking, and the protein is inserted into the membrane of the ER
• Movement of proteins between membranes

  1. Once the proteins are in the ER, they are transported by vesicles that bud off from the ER and fuse with the Golgi apparatus

  2. As proteins move through the Golgi apparatus they undergo post-translational modification

  3. The addition of carbohydrate groups is the major modification

  4. Vesicles that leave the Golgi apparatus take proteins to the plasma membrane and lysosomes

  5. Vesicles move along microtubules to other membranes and fuse with them within the cell
• The secretory pathway


  1. Secreted proteins are translated in ribosomes on the RER and enter its lumen

  2. The proteins move through the Golgi apparatus and are then packaged into secretory vesicles

  3. These vesicles move to and fuse with the plasma membrane, releasing the proteins out of the cell

  4. Many secreted proteins are synthesised as inactive precursors and require proteolytic cleavage to produce active proteins
• Field techniques used by Biologists
• Field techniques used by Biologists

  • health and safety
  • sampling of wild organisms
  • identification and taxonomy
  • monitoring populations
  • measuring and recording animal behaviour
• Ligand binding changes the conformation of a protein


  • A ligand is a substance that can bind to a protein
  • R groups not involved in protein folding can allow binding to ligands
  • Binding sites will have complementary shape and chemistry to the ligand
  • As a ligand binds to a protein-binding site the conformation of the protein changes
  • This change in conformation causes a functional change in the protein
  • Allosteric interactions occur between spatially distinct sites
  • Many allosteric proteins consist of multiple subunits (have quaternary structure)
• Protein structure


  • Stabilised by interactions between R groups: hydrophobic interactions; ionic bonds; London dispersion forces; hydrogen bonds; disulfide bridges
• Quaternary structure


  Exists in proteins with two or more connected polypeptide subunits
• Prosthetic group


  A non-protein unit tightly bound to a protein and necessary for its function
• Organisms and Evolution


  • Field techniques used by Biologists
  • Organisms
  • Variation and sexual reproduction
  • Sex and behaviour
  • Parasitism
• Interactions of the R groups

  Can be influenced by temperature and pH
• Ligand binding


  Changes the conformation of a protein
• Sampling


  • Unbiased
  • Representative
  • Number of samples and detail
  • Accuracy and Precision
  • Pilot studies and early observations
  • Equipment
  • Quantitative and Qualitative
  • Identifying the sample
• Health and Safety


  Considerations regarding hazards and risks in fieldwork
• Health and Safety considerations

  • Terrain
  • Weather conditions
  • Isolation
• Woodland Sampling


  1. Ensure pupils are aware of others around them

  2. Ensure pupils do not throw items over their shoulder or behind them

  3. Experienced member of staff on site

  4. Suitable clothing and footwear

  5. Possible use of insect repellent

  6. Aware of branches to avoid being hit in the eye

  7. Do not pull back branches

  8. Walk in single file

  9. Wear light coloured clothes

  10. Possible use of sunscreen
• Ligand


  A substance that can bind to a protein
• R groups not involved in protein folding can allow binding to ligands</b>
• Binding sites will have complementary shape and chemistry to the ligand
• Water Sampling


  1. Suitable clothing and footwear

  2. Closer supervision required whilst at the edge of the water

  3. Choose a suitable area

  4. Split the groups into smaller groups
• Quadrats


  A frame laid down to directly count or estimate percentage cover of organisms
• As a ligand binds to a protein-binding site


  The conformation of the protein changes
• Randomised Sampling using Quadrats


  1. Quadrats are often thrown to try and get a random sample

  2. Use a random number generator or random number table to select quadrat areas

  3. Use random walk to generate a random direction and distance
• Change in conformation


  Causes a functional change in the protein
• Allosteric interactions


  Occur between spatially distinct sites
• Many allosteric proteins consist of multiple subunits (have quaternary structure)
• Systematic Sampling using Line Transects


  A line is laid out and observations are recorded at specific points along the line
• Systematic Sampling using Belt Transects


  Two parallel lines are used and the plants found between the lines are recorded at given points

  
• Stratified Sampling


  Looking at the ecosystem within different strata (layers) at each sample point

  
• Allosteric proteins with multiple subunits


  Show co-operativity in binding, in which changes in binding at one subunit alter the affinity of the remaining subunits
• Allosteric enzymes


  Contain a second type of site, called an allosteric site