Unit 1 Flashcards
What is a hazard?
A hazard is anything that poses a potential threat to an individual or the environment.
What is a risk?
A risk is the likelihood of harm arising from exposure to a hazard.
What are examples of hazards in the lab?
Toxic and corrosive chemicals, heat or flammable substances, pathogen organisms and mechanical equipment.
When should a risk assessment be carried out?
Before any practical work in the lab.
What do risk assessments involve?
Identifying hazards, risks and control measures to minimise risk.
What do control measures include?
Using protective handling techniques, protective clothing and equipment and Aseptic Technique.
Why is Personal Protective Clothing worn?
PPE is worn to reduce the risk of harmful substances coming into contact with the body.
What should be considered when choosing apparatus for working with liquids and solutions?
Accuracy: How close the measurement is to the true value and Precision: How close the values are to each other.
When and how are linear dilutions carried out?
For small dilutions, this is achieved by using different volumes of the same stock solution combined with different volumes of a suitable solvent- Dilutions will differ by an equal interval.
When and how are log dilutions carried out?
For large dilutions, this is achieved by using successive dilutions as the new stock- Dilutions will differ by a constant proportion.
What is a standard curve used to determine?
A series of standards of known concentrations are measured and graphed. This graph can be used to determine the concentration of an unknown solution.
What are titrations used to determine and how are they carried out?
Titrations can be used to determine the concentration of an unknown solution. These are carried out using a burette to deliver a solution of known concentration to a solution of unknown concentration below.
How can titration be used to calculate the concentration of a solution?
The volume and concentration of the known solution needed to reach the endpoint of the reaction with a known volume of the other solution can be used to calculate the unknown concentration using V1xC1=V2xC2.
How are buffers used to control an experiment?
It allows the pH of the reaction to be kept constant because the addition of acid or alkali has little effect on the pH of the buffer.
What does a colorimeter measure?
A colorimeter measures the absorbance or transmission of light through a solution.
How does a colorimeter work?
A colorimeter works by passing a light beam at a specific wavelength through a cuvette containing a sample. Some light is absorbed by the sample, light transmitted is detected as an absorbance value.
Why does a colorimeter need to be calibrated and how is this done?
The correct wavelengths of light must be selected when using a colorimeter. An appropriate reference blank (e.g distilled water) is placed in the colorimeter and the calibration button is pressed. This is used to provide a baseline reading (0). This shows the absorbance when the sample is absent.
How is concentration measured using a colorimeter?
By measuring the absorbance of light by a sample we can determine the concentration of a coloured substance using suitable wavelength filters as there is a linear relationship between absorbance and concentration.
How is turbidity measured using a colorimeter?
By measuring the percentage transmission of light through a sample, we can measure turbidity. The lower the transmission the higher the turbidity.
What component parts can biological systems be separated by?
Solubility, size, shape or charge.
How does a centrifuge work?
A centrifuge spins a sample at high speed to separate the mixture based on density. The densest materials form a pellet at the bottom of the tube. Less dense components remain in the mixture.
What is paper and thin layer chromatography used for?
For separating different substances such as amino acids and sugars based on solubility in specific solvents.
How does affinity chromatography work and what does it separate?
Affinity chromatography is a separation technique that separates proteins based on their solubility. The mixture is passed through a column containing ligands complementary to target proteins. Target proteins bind, are removed from the mixture, and washed out in a final wash.
What is gel electrophoresis used to separate and how does it work?
Proteins and nucleic acids. Charged macromolecules move through an electric field applied to the gel matrix.
What does gel electrophoresis separate by?
Separates proteins based on charge and/or shape/size.
How does native gel electrophoresis work?
Proteins are not denatured before electrophoresis allowing analysis in their folded state and separation can be done based on size, shape and charge.
How does SDS-Page electrophoresis work?
Proteins are denatured prior to the procedure and they are given a uniform negative charge. This separated proteins on size alone.
What is a protein’s isoelectric point?
The IEP is the pH at which a soluble protein has no net charge and will precipitate out of the solution. Proteins can be separated based on their IEP.
How can proteins be separated using their isoelectric point?
If the solution is buffered to a specific pH, only the proteins that have an IEP of that will precipitate. Proteins can also be separated using their IEP in electrophoresis.
What are immunoassay techniques used for and how is this achieved?
To detect and identify specific proteins using stocks of antibodies with the same specificity, that recognise one antigen, known as monoclonal antibodies.
How do scientists know when a monoclonal antibody has bound to the target protein?
Antibodies are linked to a chemical label which signals when binding has occurred. The label is often a reporter enzyme producing a colour change or another signal.
How does western blotting work?
The separated proteins are transferred from the gel onto a solid medium after SDS-Page electrophoresis. The proteins are identified using specific antibodies with reporter enzymes.
What is bright field microscopy commonly used to observe?
Whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.
How does fluorescence microscopy work?
Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.
How do aseptic techniques work and what do they achieve?
Aseptic techniques eliminate unwanted microbial contaminants when culturing microorganisms or cells. This involves the sterilisation of equipment and culture media by heat or chemical means.
How can a microbial culture be started?
Microbial cultures can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients.
What does a medium for growing animal cells contain?
Mediums for growing animal cells contain growth factors from serum. These are proteins that promote cell growth and proliferation and are essential.
What are the lifetimes of primary and tumour cell lines?
In culture, primary cell lines can divide a limited number of times, where as tumour cell lines can perform unlimited division.
What does plating out a liquid micro-bacterial culture on solid media allow?
The number of colony-forming units to be counted and the density of cells to be estimated. Serial dilutions are often needed to achieve a suitable colony count.
What does a haemocytometer allow for?
an estimation of the number of cells in a liquid culture to be calculated.
How are haemocytometers used?
Estimates of viable and total cell counts can be made using haemocytometers by counting the cells in an area of the grid and calculating using the volume of the area of the grid. Trypan blue die is absorbed by dead cells so total and viable counts can be achieved.
How does vital staining work?
Vital staining is required to identify and count viable cells by adding a stain to a cell culture and observing which cells have taken up the stain.
What is the proteome?
The entire set of proteins expressed by a genome.
Why is the proteome larger than the genome?
Due to alternative RNA splicing and post-translational modification as more than one protein can be expressed from a single gene.
What do genes that don’t code for proteins?
Not all genes are expressed as proteins in a particular cell type, genes that do not code for proteins are called non-coding RNA genes and RNA molecules that control the expression of other genes.
What conditions can cause the set of proteins expressed by a cell to vary?
Metabolic activity of the cell, cellular stress, response to signalling molecules and diseased vs healthy cells.
What system do eukaryotic cells have in place to increase the total area of membrane available?
As eukaryotic cells have a small surface area to volume ratio the area is too small to carry out vital functions. They have a system of internal membranes available for cellular functions.
What does the endoplasmic reticulum form a network of?
The ER forms a network of membrane tubules continuous with the nuclear membrane.
What is the endoplasmic reticulum involved in?
The synthesis of lipids and proteins.
When is the ER referred to as the rough ER
When ribosomes are on its cytosolic face.
What is the Golgi apparatus?
A series of flattened membrane discs.
What is the Golgi apparatus involved in?
The transport and modification of proteins.
What are lysosomes?
Membrane-bound organelles containing a variety of hydrolases that digest proteins, lipids, nucleic acids and compartments.
What is the function of vesicles?
Vesicles transport materials between membrane compartments.
What ER are lipids synthesised in and where do they go afterwards?
Lipids are synthesised in the smooth ER and are inserted into the membrane.
Where does the synthesis of proteins begin?
The synthesis of all proteins begins in the cytosolic ribosomes.
Where are the cytosolic proteins transferred to after synthesis in the ribosome?
To the cytosol.
How do transmembrane proteins halt translation and where do they go?
A signal sequence halts translation and directs the ribosome to dock with the ER, forming RER. The protein is inserted into the ER membrane.
What is a signal sequence?
A short stretch of amino acids at one end of the polypeptide that determines the eventual locations of a protein in a cell.
Where do proteins go after they enter the ER?
The proteins are transported by vesicles and fuse with the Golgi apparatus.
What happens to proteins in the Golgi apparatus?
As proteins move through the Golgi apparatus they undergo post-translation modification.
What is the main modification occurring to proteins in the Golgi?
The addition of carbohydrate groups is the major modification taking place.
Where do vesicles that leave the Golgi take proteins to?
Vesicles that leave the Golgi apparatus take proteins to the plasma membrane and lysosomes.
Where are secret proteins translated?
Secreted proteins are translated in the ribosome on the RER and enter its lumen.
Where do secreted proteins go after they move through the Golgi apparatus?
Secreted proteins are packaged into secretory vesicles. These vesicles move to and fuse with the plasma membrane, releasing the proteins out of the cell.
What is protealytic cleavage?
The process of breaking peptide bonds between animism acids in proteins.
How are many secreted proteins synthesised?
Many secreted proteins are synthesised as inactive precursors and require proteolytic cleavage to produce active proteins. (stops digestive enzymes damaging cells).
What are proteins made from?
Proteins are polymers of amino acids.
What bonds are amino acids linked by?
Amino acids are linked by peptide bonds to form polypeptides.
What differs in the structure if amino acids?
Amino acids have the same basic structure only differing in the R group present.
What do amino acid R groups vary in?
Shape, size, charge, hydrogen bonding capacity and chemical reactivity.
What are the 4 classifications of R groups?
Acidic- negatively charged
Basic- positively charged
Polar
Hydrophobic- non polar
What determines the function of a protein?
The wide variety of functions carried out by proteins results from diversity of R groups.
What is the primary structure of a protein?
The primary structure is simply the sequence in which the amino acids are synthesiser into a polypeptide.
What is the secondary structure in a protein?
Hydrogen bonding along the backbone of the protein strands results in regions of secondary structure.
What are the 3 types of secondary structure?
A-helix, parallel or anti-parallel beta-sheet and turns.
What is the tertiary structure of a protein?
Tertiary structure involved the folding of the polypeptide chains to give a more complex 3D structure.
What interactions between R groups stabilises the conformation if a tertiary structure?
Hydrophobic interactions, ionic bonds, LDFs, hydrogen bonds and disulphide bridges.
What are disulphide bridges?
Disulphide bridges are covalent bonds formed between R-groups containing sulphur.
What is quaternary structure of a protein?
Quaternary structure exists in proteins with two or more connected polypeptide subunits. (This describes the a partial arrangement of subunits).
What is a prosthetic group?
Prosthetic groups are non protein unit tightly bound to a protein and necessary for its function. (eg haem for binding of oxygen to haemoglobin)
How does increasing temperature effect interactions if the R groups?
Increasing temperature disrupts interactions that hold the protein in shape. The protein unfolds and becomes denatured.
How does pH effect the charges of the R groups?
The charges on acidic and basic R groups are affected by pH. As H increases or decreases from from the optimum, the normal ionic interaction between charged groups are lost. This changes the shape of the protein.
What is a ligand?
A ligand is a substrate that can bind to a protein.
What R-groups allow binding to ligands?
R-groups not involved in protein folding can allow binding to ligands.
What do binding sites have that is complimentary to the ligand?
Binding sites will have complementary shape and chemistry to the ligand.
