Laboratory techniques for biologists Flashcards
Tools to measure quantities accurately.
Scales
Measuring cylinders
Pipettes
Auto-pipettes
Why are dilutions used?
So a measurable value can be obtained.
Explain a LINEAR dilution series.
A range of dilutions differing by an equal interval (e.g: 0.1, 0.2, 0.3…).
Each concentration is made individually.
Explain a LOG dilution series.
A range of dilutions differing by a constant proportion.
The subsequent solution is made from diluting the solution before.
Earlier measurement errors are compounded in later dilutions.
Explain some of the things which a colorimeter would measure.
Measures the: Concentration of a pigment in a solution
Turbidity of a liquid
Density of cells in a culture
How does a colorimeter work?
Measures how much light is absorbed by a sample on a small transparent cuvette.
A denser sample shows a greater degree of transmission.
Explain a method used for a colorimeter which acts as a control.
The machine is calibrated using a blank cuvette which contains the solvent only.
Common uses of a colorimeter.
Measure the density of a cell culture growing in a liquid broth.
Measure the concentration of betanin leaking from denatured beetroot cells.
Measure the rate of reaction of enzymes like dopa oxidase.
Quantify the results of an ELISA demonstration.
How would you determine the concentration of a substance which has an unknown substance?
Plot the absorbance reading for a series of known concentrations of a substance/culture.
The line produced on the graph can be used as a reference for unknown concentrations of the same substance/culture.
The term which allows you to identify an estimate of the concentration of a substance.
Interpolation.
What is a buffer?
Aqueous solutions that show very little variation in pH.
How would you control the pH of a solution?
Using a buffer.
Give an example(s) of buffers in mammalian blood.
Carbonic acid
Bicarbonate anions
What is centrifugation?
The method of separating materials in suspension according to their density.
Describe the process of centrifugation acting on a cell sample?
Various constituent cellular structures and molecules are separated and can be studied in isolation.
Describe the range in rpm which material is rotated at.
2000-120000 rpm.
Where is material placed when it undergoes centrifugation.
A centrifuge tube.
What causes the constituents to separate.
G-force.
What is the name of the material which forms at the bottom of the centrifuge tube created by the denser items in a sample.
A pellet.
What is the liquid fraction above the pellet in a centrifuge tube called?
A supernatant.
What are the typical uses for centrifugation?
Enzyme extraction from tissues
Study of electron transport chain activity(kill reaction)
Types of chromatography.
Paper Chromatography
Thin Layer Chromatography.
Describe the use of chromatography.
The separation of amino acids according to their solubility.
What results in different rates of movement of substances in chromatography.
Different relative affinities of materials for the solvent.
Which part of a chromatography set up is hydrophilic?
The paper.
Which part of a chromatography set up is hydrophobic?
The solvent.
What is affinity chromatography?
The technique used to separate a specific protein from a mixture.
How are antibodies/ligands specific to proteins immobilised?
Form in agarose gels and packed into a column.
Explain how specific proteins are separated from a mixture in affinity chromatography.
The protein mixture is poured through the column and only the protein interested in binding to the antibody/ligand binds.
Other proteins don’t bind and can be washed away.
How would you recover the target protein in affinity chromatography?
Wash the column with a buffer(which lowers the affinity of the protein to the ligand) or a solution containing free ligand.
How does protein electrophoresis work?
The process uses the current flowing through a buffer to separate proteins depending on their size and charge.
Does the protein running through electrophoresis need to be non-denatured or denatured?
No.
Non-denatured = native
Denatured = non-native
How would you denature a protein?
With the presence of heat or detergent.
What happens when a protein is denatured.
The chain unfolds.
What does unfolding a protein do to its charge?
Results in a linear protein with a uniform charge along its length.
How does unfolding a protein affect its rate of migration?
Rate of migration depends only on size(rather than charge and size).
What is an isoelectric point?
The pH at which a protein has an overall neutral charge.
How does a pH higher or lower than the isoelectric point affect the charge of a protein.
Causes the surface of the protein to be charged making it soluble.
How is a protein soluble?
Water molecules interact with the proteins charge keeping it suspended in solution.
What happens when the charge of the protein is neutral?
A solid precipitate forms out of the solution.
How can you separate multiple different proteins in a mixture.
Slowly change the pH of the solution so different proteins become precipitates and can be extracted at their
different isoelectric points.
What are antibody techniques used for
Detection and identification of specific proteins
What are antibodies
Y-shaped globular proteins that bind to antigens
Where and why are antibodies produced
Produced by b-lymphocytes as an immune response
Where are b-lymphocytes produced
Spleen/bone-marrow
Describe polyclonal antibodies
Many different antibodies attacking the same antigen
Describe monoclonal antibodies
A supply of identical antibodies that will bind to the same feature of an antigen
How do b-lymphocytes divide in culture
They are fused with myeloma cells from an immortal cell line using PEG.
What is the name given to fused b-lymphocyte/myeloma cells
hybridomas
How is the an antibody extracted and purified
Centrifugation
What are monoclonal antibodies used for
Diagnosis and detection of disease
How do immunoassay techniques work
Reporter enzyme linked to a monoclonal antibody produces a coloured product when a specific protein binds
What is fluorescent-labeled protein blotting
A technique for identifying specific proteins that have been separated using gel electrophoresis.
Explain how fluorescent-labeled protein blotting works
Proteins are blotted onto a nitrocellulose membrane to record their positions easily, the filter is flooded with fluorescent-labelled monoclonal antibodies which detect and identify the different proteins.
Define histochemistry
The study of tissues using stains and histochemistry
What is Fluorescent-immunohistochemistry used for
To visualise the distribution of specific cellular components in living cells
Describe the growth mediums in cell cultures
Grown in suspension in a liquid medium
Grown on/within a solid medium
Describe an inoculum
Growing cells are added to a culture from a previous culture (cells can be enzymatically removed or an explant may be taken)
What are aseptic techniques used for
To prevent contamination by using sterile materials and appropriate treatments of sources
Give some examples of aseptic techniques
Pass the neck of a bottle through a bunsen, pass the loop through a bunsen
What is a growth factor
A complex medium which allow rapid cell division.
Consists of proteins, salts, vitamins, glucose, maybe antibiotics.
How can growth factors be added to a culture
Via an animal serum
Describe the pathway of cell culture
Cells are removed by proteolytic enzymes which adhere to the surface of the flask.
They then divide and form a monolayer until the nutrients are used up.
To keep the culture alive they must be transfered to a culture with more growth factors.
What is the hayflick limit
The amount of cell divisions a culture can withstand before it dies (about 60)
What is unique about myeloma (cancer cells)
The Hayflick limit doesn’t exist and the cell lines are immortal
Define ‘totipotent’
Plant cell that is able to differentiate into all cell types to form a new organism
Define explant
Small pieces of plant tissue that can be placed on a solid medium
What are growth regulators used for
To induce embryogenesis to generate new embryonic plants
What are haemocytometers used for
To count cell density through a known volume of medium
What is a total and viable cell count
Total - All cells
Viable - Living cells (found by applying a stain to distinguish living cells)
Define genome
The complete set of DNA
Define proteome
The entire set of proteins that can be expressed from the genome
Why is the proteome larger than the genome
Alternative RNA splicing
Post-translational modification
What does an amino acid consist of
A carbon with a hydrogen, a carboxylic acid group, an amine group and an R group
What are the classes of R groups
Acidic (COOH (negatively charged))
Basic (NH2 (positively charged))
Polar (OH)
Hydrophobic (hydrocarbon)
