Cells And Protiens Flashcards
Hazards in the lab include
Toxic or corrosive chemicals
Heat or flammable substances
Pathogenic organisms
Mechanical equipment
We can identify and control measures to minimise risks and reduce hazards by creating a…..
Risk assessment
Risk
The likely hood of harm arising from the exposure to a hazard
Control measure
Using appropriate handling techniques, protective clothing and equipment and aseptic techniques
Linear dilutions
Dilutions differ by an equal interval, for example: 0.1, 0.2, 0.3 ect
Log (serial) dilutions
Differ by a constant proportion, for example 10^-1, 10^-2, 10^-3 ect
Standard curve
Some investigations need you to plot known measurements on a graph to then use to determine unknown values
Buffers
A solution where adding acids or alkalis have very small effects on the ph. This allows ph in a reaction mixture to be kept constant
Colorimeters can be used to quantify….
Concentration and turbidity
Before use the colorimeters need to be…
Calibrated to provide a baseline reading
Centrifuge
Samples are spun at increasingly high speeds, sometimes up to 18,000rpm
This separates substances according to density
More dense components settle to form the pellet whilst less dense components remain in the supernatant
Paper and thin layer chromatography
Used to separate and identify substances such as amino acids and sugars
The speed that the solute travels along the chromatograph depends on its differing solubility in the solute used
Affinity chromatography
Used to separate proteins
A solid matrix or gel column is created with specific molecules (usually receptors) bound to it
Soluble target proteins in a mixture, with a high affinity for these molecules become attached to them as the mixture passes down the column
Non target molecules are washed out
Gel electrophoresis
Used to separate proteins and nucleic acids
The samples are backed into hells which have an electric current running through
Charged molecules will move towards the opposing charge
Smaller molecules travel faster and further than larger molecules
Native gels
Separate proteins by shape, size and charge and ensure they do not denatured the molecule
SDS-PAGE
Separates proteins by size by giving all molecules an equally negative charge and denaturing them
Isoelectric point (IEP)
Used to separate proteins from a mixture
IEP is the specific ph at which a soluble protein has no net charge and will precipitate out of a solution
The solution is buffered to a specific PH, only the proteins that have an IEP of that PH will precipitate
This can be used along with electrophoresis as a proteins stops migrating through the gel at its IEP
Immunoassay
Used to detect and identify proteins
Uses antibodies with the same specificity known as monoclonal antibodies an antibody specific to the protein antigen linked to a chemical label (or fluorescence or chemiluminescence)
The label is often a reporter enzyme producing a colour change
Can be used to detect diseases
ELISA Techniques
Enzyme-linked Immunoabsorbent Assay is an analytical technique using antibodies to detect the presence of an antibody within a soloution
There are thre types, direct, in direct and sandwich
Direct ELISA
The antigen is allowed to bind to the surface of a multi well plate
A primary antibody linked to a reporter enzyme is added to the well and binds to the antigen
Indirect ELISA
The antigen is allowed to bind to the surface of a multi well plate
A primary antibody is added to the well and binds to the antigen.
A secondary antibody linked to a reporter enzyme is added and binds to the antigen
Sandwich ELISA
A capture antibody is allowed to bind to the surface of a multi well plate
The antigen is added and allowed to bind to the capture antibody
A primary antibody binds to the antigen
A secondary antibody linked to a reporter enzyme is added to the well and binds to the primary antibody
Western Blotting
Used after SDS-PAGE Electrophoresis
Once the proteins are separated they are transferred or blotted onto a solid medium
Proteins can be identified by ELISA
Bright field microscopy
Used to observe whole organisms, parts of organisms, thin sections, dissected tissue or individual cells