UNIT 1 - KA1 Flashcards
What is a hazard
A hazard is a source of potential harm
What are hazards in the lab (4)
toxic, or corrosive chemicals
heat or flammable substances
Pathogenic organisms
Mechanical equipment
What is a risk
Risk is the likelihood of harm arising from exposure to a hazard
Risk assessment
Risk assessment involves identifying control measures to minimise the risk
Control measures
Appropriate handling techniques
Protective clothing and equipment
Aseptic technique
What is a ph buffer
A ph buffer is a solution whose pH changes very little when a small amount of acid or base is added to it
How do buffers work
Buffers work by allowing the addition of hydrogen or hydroxide ions without affecting the pH of the solution
Why are buffer solutions used
Buffer solutions are used as a means of keeping ph at a nearly constant value
What is the application of centrifugation
Separation according to density
How does a centrifuge separate substances of differing density
More dense compounds settle in the pallet whereas less dense components remain in the supernatant
What are the names given to the solid and liquid in a centrifuge tube
The solid found at the base of the tube is called the pellet and the liquid the supernatant
What are linear dilutions used for
Used if you need a sample over a reasonably small range
How do dilutions in a linear dilution series differ
Dilutions in a linear dilution series differ by an equal interval for example 0.1, 0.2 ,0.3 and so on.
What are serial (log dilutions) used for
Where dilution is needed across a wide range a serial dilution produces sampled which differ from the next by an order of magnitude each time
How do dilutions in a log dilution series differ
Dilutions in a log dilution series differ by a constant proportion, eg 10^-1 , 10^-2 , 10^-3 and so on
How does the production of a standard curve determine an unknown
Plotting measured values for known concentrations to produce a line or a curve allows the concentration of an unknown to be determined from the standard curve
What can paper and thin layer chromatography be used for
Paper and thin layer chromatography can be used for separating different substances such as amino acids and sugars
What does the speed at which the solute travels through the chromatogram depend on
The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used
How is a solid matrix or gel column created
A solid matrix or gel column is created with specific molecules bound to the matrix or gel
What happens to soluble target proteins in affinity chromatography
Soluble target proteins in a mixture with high affinity for these molecules become attached to them as the mixture passes down the column
What happens to non target molecules in affinity chromatography
Other non - target molecules with a weaker affinity are washed out
What is the process of electrophoresis
Electrophoresis is the process of moving charged molecules in solution by applying an electric field across the mixture
What three factors determine the speed at which molecules move in protein electrophoresis
The molecules move with a speed dependant on their charge, shape size and so they can be separated from other molecules using this technique
What is the gel in gel electrophoresis made of
The gel is made of agarose
How is the tank set up in gel electrophoresis
The tank is set up with a positive and negative terminal and an electric current is applied
Where do the fragments move towards in gel electrophoresis
The fragments move towards the electrode which is of the opposite charge to the fragments
Where do smaller and larger fragments move to
Smaller fragments move more Quickly (do not get tangled on the agarose protein strands) than the larger fragments.
How do native gels separate proteins
Native gels separate proteins by their shape, size and charge
How do native gels separate by shape size and charge
Native gels do not denature the molecule so that separation is by shape, size and charge.
How does SDS page separate proteins
SDS page gives all the molecules an equally negative charge and denatures them separating proteins by size alone
What is the iso electric point of a soluble protein
IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
What happens to proteins 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
What is another way soluble proteins can be separated
Soluble proteins can be separated using an electric field and a pH gradient.
When does a protein stop migrating through the gel
A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
What are antibodies
Antibodies are y shaped globular proteins produced by B lymphocytes as part of the specific immune response
What types of cells produce antibodies
Each b lymphocytes produces one specific antibody that binds to one specific type of antigen
What does the binding of antibody’s and antigens render
The binding renders the antigen harmless and promotes destruction of invading pathogens
What are immunoassay techniques used for
Immunoassay techniques are used to detect and identify specific proteins
What type of antibodies do immunoassay techniques use
These techniques use stocks of antibodies with the same specificity known as monoclonal antibodies
What should any antibody in an immunoassay be linked to
Any antibody used in an immunoassay must be linked to a detective label to allow scientists to detect when binding has occurred
What is a polyclonal antibody
Many different antibodies will have formed to different parts of the same antigen. Each antibody is made by a single B lymphocyte clone. A serum made in this way with many different antibodies against the same antigen is described as polyclonal
What are monoclonal antibodies
These are antibodies which are identical to each other and will bind to exactly the same feature (eg same protein) of the antigen
How should b lymphocytes be grown in relation to monoclonal antibodies
A single line of B lymphocytes must be grown each secreting the same specific antibody
What forms are the detectable label in
These labels may be in the form of a reporter enzyme which causes a colour change in the presence of a specific antigen. Chemiluminescence, fluorescence and other reporters can also be used
In some cases what could the assay do
In some cases the assay uses a specific antigen to detect the presence of antibodies.
What is an immunoassay
An immunoassay is a biochemical test that measures the presence of concentration of a macromolecule in a solution through the use of an antibody
What is one of the main types of immunoassay
One of the main types of immunoassay carried out is the Elisa test
What happens in the Elisa test
In this test antibodies linked to reporter enzymes cause a colour change in the presence of a specific antigen
Draw a label of detection of a protein or antibody
Check jotter
What is immunohistochemical staining
Labelled antibodies are also used for immunohistochemical staining. Where an antibody to a particular antigen can be used to identify tissue proteins
What is western blotting
Western blotting is a technique used after SDS page electrophoresis. The separated proteins from the gel are transferred onto a solid medium
What is the advantage of immunohistochemistry
The advantage of immunohistochemistry lies in the fact that it is capable of showing exactly where a certain protein is being expressed within a tissue sample
What is the first stage of western blotting
1 - SDS page electrophoresis is used to separate proteins in the extract ( separated according to size)
What is the second stage of western blotting
2- the proteins are then transferred to a membrane where they are stained with antibodies. The antibodies bind where the target protein is present
What is the third stage of western blotting
3 - the membrane is probed with an antibody specific for the protein of interest. It binds where the protein of interest is present
What is the fourth stage of western blotting
4- stained blot is transferred and fixed to autoradiograph visualise antigen binds that have been stained
What is bright field microscopy used for
Bright-field microscopy is commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
What is the features of fluorescence microscopy
Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
What is cell structure
Cell structure is the ability to grow cells in an artificial laboratory environment
What do aseptic techniques eliminate
Aseptic technique eliminates unwanted microbial contaminants when culturing micro- organisms or cells
What do aseptic techniques 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 type of culture media exists
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 in animal cells
Growth factors are proteins that promote cell growth and proliferation.
What are growth factors essential for
Growth factors are essential for the culture of most animal cells.
What is the difference between primary cell lines and tumour cell lines
In culture, primary cell lines can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions
What is the benefit of plaiting out 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
What is serial dilution
Serial dilution is often needed to achieve a suitable colony count
What is a haemocytometer used for
A hameocytometer may be used to estimate cell numbers in a liquid culture
What are the steps for cell counting technique question
- Count the number of cells in the central square
- Work out the volume of each square (lxbxh)
- Multiply up to find the number of cells which there would be in 1cm3
Why is vital staining required
Vital staining is required to identify and count viable cells (only stains dead or living)
What is a primary cell line
A primary cell line refers to cells that have been isolated and then used immediately. However most animal cells can only perform a finite number of divisions before the cell line can no longer be maintained
Tumour cell lines
Tumour cell lines can preform an unlimited number of divisions and so the sub culture can be maintained indefinitely
