Cells And Proteins Flashcards
Describe a hazard and the types of hazards
Hazards in the lab include toxic or corrosive substances, heat or flammable substances, pathogenic organisms and mechanical equipment
Describe what a risk is
Risk is the likelihood of harm arising from exposure to a hazard
Describe risk assessment
Risk assessment involves identifying control measures to minimise risk
Name these control measures
Control measures include using appropriate handling techniques, protective clothing and equipment, and aseptic technique
Describe linear and log dilutions
Dilution in a linear dilution series differ by an equal interval e.g 0.1, 0.2, 0.3 and so on.
Dilutions in a log dilution series differ by constant proportion e.g 10^-1, 10^-2, 10^-3 and so on.
Production of a standard curve to determine an unknown, and what is a standard curve
A standard curve is used to determine the concentration of a solution.plotting measured values for known concentrations to produce a line or curve allows concentration of an unk own to be determined from the standard curve.
Describe the use of buffers to control Ph
PH is a measure of the acidity or basicity of a solution acids<7 neutral=0 bases>7.A buffer is a solution whose pH changes very little when a small amount of acid or base is added to it.Addition of acid or alkali has a very small effect on the pH of a buffer, allowing the pH of a reaction mixture to be kept constant.
Describe the methods and uses of a colorimeter to quantify concentration and turbidity
A colorimeter is used to measure the concentration of a coloured solution or the turbidity(cloudiness).Calibration with appropriate blank(solvent), use of absorbance to determine concentration of a coloured solution using suitable wavelength filters, use of percentage transmission to determine turbidity, such as cells in suspension.A colorimeter works by passing a light beam at a specific wavelength, though a cuvette containing a sample solution.some of the light is absorbed by the sample therefore light of a lower intensity hits the detector.for turbidity a denser sample will show a lower degree of transmission.
Describe the Use of centrifuge to separate substances of differing density
Centrifugation allows substances to be separated according to their destiny. More dense components settle in the pellet; less dense components remain in the supernatant.
Describe paper and thin-layer chromatography and affinity chromatography
Paper and thin layer chromatography can be used for separating different substances such as amino acids and sugars. The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.
Principle of affinity chromatography and its use in separating proteins
A solid matrix or gel column is created with specific molecules bound to the matrix or gel. Soluble, target proteins in a mixture, with a high affinity for these molecules, become attached to them as the mixture passes down the column. Other non-target molecules with a weaker affinity are washed out.
Principle of gel electrophoresis and its use in separating proteins and nucleic acids
Charged macromolecules move through an electric field applied to a gel matrix
Describe how native gels separate proteins
Native gels separate proteins by their shape, size and charge
Decribe wether native gels denature and how they separate proteins
Native gels do not denature the molecule so that separation is by shape, size and charge
Describe how SDS-PAGE separates proteins and what they do
SDS–PAGE separates proteins by size alone. SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.
Describe how proteins can be seperated
Proteins can be separated from a mixture using their isoelectric points (IEPs)
Describe what isoelectric point is
IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
Describe how proteins will precipitate if a solution is buffered to a specific pH
If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate
Describe how an IEP can be used in electrophoresis
Proteins can also be separated using their IEPs in electrophoresis
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Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
What techniques are used to identify proteins
Immunoassay techniques are used to detect and identify specific proteins
What do immunoassay techniques use
These techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies
How are antibody specific to a protein antigen linked
An antibody specific to the protein antigen is linked to a chemical ‘label’
Describe the label and other reporter enzymes
The ‘label’ is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used.
What else can the assay use to detect antibodies
In some cases the assay uses a specific antigen to detect the presence of antibodies.
What is western blotting and when is it used
Western blotting is a technique, used after SDS–PAGE electrophoresis
The separated proteins from the gel are transferred (blotted) onto a solid medium.used for identifying specific proteins that have been separated using SDS-PAGE gel electrophoresis.
How can proteins be identified
The proteins can be identified using specific antibodies that have reporter enzymes attached
Describe what bright-field microscopy is
Bright-field microscopy is commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
Describe what fluorescence microscopy is
Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
Describe what aseptic technique is
Aseptic technique eliminates unwanted microbial contaminants when culturing micro- organisms or cells
What does aseptic technique involve
Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.
How can a microbial culture be started
A microbial culture can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients
What is does culture media promote
Many culture media exist that promote the growth of specific types of cells and microbes.
How are animal cells grown
Animal cells are grown in medium containing growth factors from serum
What are growth factors and their importance
Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells
Describe in terms of culture primary cell lines and tumour cell lines and division
In culture, primary cell lines can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions
Explain the importance of plating out of a liquid microbial culture
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
Why is serial dilation needed for colony count
Serial dilution is often needed to achieve a suitable colony count
What is haemocytometry used for
Method and use of haemocytometer to estimate cell numbers in a liquid culture
What is vitale staining used for
Vital staining is required to identify and count viable cells
What is the proteome
The proteome is the entire set of proteins expressed by a genome
