Unit 1 - Cells and Proteins Flashcards
Name 2 things which can be intrinsically harmful when working in a lab.
Chemicals
Organisms
Who, or what, is at risk when working in a lab?
People working in the lab
Other organisms
The environment
In reference to control measures, what is elimination?
Replace the hazardous substance with a less harmful equivalent or remove a step
In reference to control measures, what is substitution?
Some hazards may be very particular to the chemical/organism involved and it may be possible to use alternatives
In reference to control measures, what is isolation?
Carry out the procedure in a contained environment
In reference to control measures, what is education?
Train people to follow standard methods of practise which reduce risk
In reference to control measures, what is personal protective equipment?
Safety equipment
Which control method is most preferred and which is least preferred?
Most preferred- elimination
Least preferred- personal protective equipment
What is the purpose of a risk assessment?
Risk assessments are used to keep workers, other organisms and the environment safe, through the use of control methods.
What is the use of a burette?
Used for titrations. Can make accurate measurements of small volumes (1-100 cm^3)
What is the use of a Pipette?
Best used to measure volumes from 30 micro-litres to 2 cm^3. Less accurate.
What is the use of an Auto Pipette?
Allows small volumes of liquid to be measured very accurately.
What is the use of a syringe?
Can be used to measure from 0.5 micro-litres to 50 cm^3, very accurate If used correctly
What is dilution?
Decreasing the concentration of a solution while adding another substance.
What is the formula for calculating concentration in dilution?
C1V1 = C2V2
What is Log Dilution and when is it used?
Involves dilution by 1/10th each time, useful when culturing microbes.
What is a standard curve?
A graph of known concentrations that allows you to determine unknown concentrations.
How can pH be controlled in an experiment?
pH can be controlled in an experiment by adding a buffer solution.
How can pH be tested?
pH can be tested with a pH metre or universal indicator and a colour chart.
What is centrifugation?
Centrifugation is a process used to separate substances by their density. As the machine spins, a solid pellet forms at the bottom of the vessel.
What is the supernatant?
The supernatant is the remaining liquid left during the process of centrifugation.
Name 3 types of chromatography.
Paper Chromatography
Thin-layer Chromatography
Affinity Chromatography
What can chromatography be used to separate?
Chromatography can be used to separate:
- Amino acids
- Proteins
What is responsible for the separation of molecules in chromatography?
Solubility is responsible for the separation of molecules in paper and thin-layer chromatography. While Affinity is responsible for separation in affinity chromatography.
What is electrophoresis?
In electrophoresis, proteins are put in a gel with different charges at either end, the resulting movement of the proteins differ (due to their different charges).
What is the main factor affecting the movement of proteins in electrophoresis?
Molecular size is the main factor affecting the movement of proteins.
What is the isoelectric point?
The isoelectric point of a protein is the pH at which it has an overall neutral charge.
What happens to a protein when it reaches its isoelectric point?
At the isoelectric point, the overall neutral charge of the protein allows it to form a solid and precipitate out of solution.
What are antibody techniques used to detect?
Antibody techniques are used for the detection of specific proteins.
What is immunoassay?
Immunoassay is a biochemical test based on antigen binding principals, that is used for detecting specific proteins.
What is meant by antigen binding principals?
The specificity of antibodies and their ability to recognize and bind with only one antigen.
What is ELISA?
ELISA (enzyme-linked immunosorbent assay) is an analytical technique which uses antibodies to detect the presence of an antigen within a solution.
What are the three forms of ELISA?
Direct, Indirect and Sandwich.
How can antibodies be labelled for detection?
Reporter enzymes catalyse a colour change reaction that is used to detect and quantify the presence of a specific antigen.
How does Protein Blotting work?
A mixture of proteins is extracted.
The protein mix is separated using Gel Electrophoresis.
The Gel is placed underneath a membrane filter and overlaid with absorbent paper.
A current is applied and the proteins migrate onto the filter.
A label for the protein of interest is added.
The protein of interest can now be visualised.
What is immunohistochemical staining used for?
Immunohistochemical staining is a technique that is used to visualise the distribution of specific cellular components in live cells.
What is a monoclonal antibody?
A monoclonal antibody is a supply of antibodies that are identical and will bind to exactly the same feature of the antigen.
What are the two types of cells fused when producing monoclonal antibodies?
B Lymphocytes and Myeloma cells are fused when producing monoclonal antibodies.
Why are B Lymphocytes used when producing monoclonal antibodies?
B Lymphocytes are used as they allow a specific antigen to be targeted.
Why are Myeloma cells used when producing monoclonal antibodies?
Myeloma cells are used since B Lymphocytes do not divide in culture, they allow duplicate cells to be produced.
What is the name of the cell produced when B Lymphocytes and Myeloma cells are fused?
A Hybridoma.
What is the technique used to fuse B Lymphocytes and Myeloma cells?
The technique used for the fusion of the two cells is Polyethylene Glycol (PEG).
What is a Bright Field Microscope?
A microscope used to examine whole organisms, parts of organisms or thin sections of tissue.
What is a Fluorescence Microscope?
A microscope used to detect specific proteins that have been fluorescently labelled antibodies.
What is Aseptic technique?
Aseptic technique is the procedure carried out to ensure sterile conditions.
What is an Inoculum?
The inoculum is the original stack of cells.
What is an explant?
Explants are small cuttings of whole tissue.
What is the piece of equipment used to calculate cell counts?
A haemocytometer is used to calculate cell counts.
What is the purpose of staining when making cell counts?
The purpose of staining when making cell counts is so you can see the cells.
What is vital staining and how does it work?
Vital staining is a method of staining where only the dead cells change colour. This allows you to distinguish between dead and living cells. Both types of cells would take up the dye but the living cells would pump it back out.
What is the viable cell count?
The total number of living cells in the sample.
What is the Non-viable cell count?
The total number of dead cells in the sample
What is the total cell count?
The total number of cells both living and dead in the sample.
How would you perform a cell count using a haemocytometer?
Work out the volume under the grid.
Divide 1 by the volume to find the number of times the volume goes into 1cm^3.
Count the cells under the grid.
Multiply the number of cells by the times the volume goes into 1cm^3 to find the cells concentration.
What does a Simple culture medium contain?
A simple culture medium allows conditions for gas exchange, has a suitable pH and temperature and has a suitable growing surface.
What is the purpose of a simple culture medium?
The purpose of a simple culture/growth medium is to provide the basic requirements for cell growth.
What does a complex growth medium contain?
Growth factors - stimulate proliferation (division) of cells.
Serum - Source of growth factors
Food source e.g. Glucose - Provides energy for proliferation.
pH buffer - Prevents changes in pH.
Auxins - Plant growth regulator.
Why is serum added to media when culturing animal cells?
Serum is added to media when culturing animal cells as it is a source of growth factors meaning it stimulates proliferation of cells.
What is a monolayer?
A monolayer is a single-cell, thick confluent layer of cells.
Why is the lifetime of primary cells shorter than that of cancer cells?
Cancer cells divide infinitely as they don’t have normal controls.
Why are auxins added to media when culturing plant cells?
Auxins regulate plant growth, this allows the plants to grow in the most energy absorbing way.
What is the proteome?
The proteome is the entire set of proteins expressed by the genome.
What is the genome?
The genome is the entire hereditary information encoded in DNA.
What are the two processes responsible for the proteome being much larger than the genome?
Alternative RNA splicing and post-translational modification are responsible for the proteome being much larger than the genome.
Why is the entire proteome not expressed in all cells?
The entire proteome is not expressed in all cells due to regulation of gene expression.
Name the monomer that forms the basic structure of a protein.
Amino acids are the monomers that form the basic structure of a protein.
What is the polymer formed by amino acids when making proteins?
The polymer formed is a polypeptide, this is the primary level of protein structure.
What are the bonds involved in the basic structure of a protein and how are they formed?
Peptide bonds are involved in the basic structure of a protein.
These are formed by dehydration reactions (between a carboxyl group and an amide group.
What are the four main classes of amino acid side chains (R groups)?
Acidic
Basic
Polar
Non-Polar
For Acidic R groups;
Give an example of an amino acid
Name the type of bonding
Give a common feature
Acidic R groups are negatively charged.
Example of an amino acid is Aspartic acid.
Contains hydrophilic bonding.
Have a carboxyl group which ionises to make them acidic.
For Basic R groups;
Give an example of an amino acid
Name the type of bonding
Give a common feature
Basic R groups are positively charged.
Example of an amino acid is Arginine.
Contains hydrophilic bonding.
Have an additional amino group which ionises to NH3+.
For Polar R groups;
Give an example of an amino acid
Name the type of bonding
Give a common feature
Example of an amino acid is Glutamine.
Contains hydrophilic bonding.
Different functional groups which contain -OH, -SH or =O.
For Non-Polar R groups;
Give an example of an amino acid
Name the type of bonding
Give a common feature
Example of an amino acid is Glycine.
Contains hydrophobic bonding.
R-group is a hydrocarbon.
What is the secondary structure of a protein?
The secondary structure of protein contains three regular Sub-Structures; Alpha Helix, Beta Sheets, and turns.
What is an Alpha Helix?
The protein is a helix shape held together by hydrogen bonds between amino acids.
What are Beta sheets?
Parts of the polypeptide chain run alongside each other to form a corrugated sheet.
Antiparallel sheets have horizontal hydrogen bonds.
Parallel beta sheets have diagonal hydrogen bonds.
What are turns?
Reverses the direction of the polypeptide.
What is the type of bond involved in holding the secondary structure of proteins in place?
Hydrogen bond.
What is the tertiary structure of a protein?
The tertiary structure is the three-dimensional shape of a protein. This further folding is due to the interactions between R-Groups.
Name the types of bonds that can hold the tertiary structure in place.
Hydrophobic interactions Ionic Bonds Hydrogen Bonds Van Der Waals interactions Disulfide bridges.
What are Non-protein parts added to a protein?
Prosthetic groups.
Give two examples of prosthetic groups.
Carbohydrates
Nucleic acid.
Describe a proteins quaternary structure.
Quaternary structure exists in proteins that have two or more tertiary sub-units joined together. The bonding between sub-units means that changes to the conformational shape of one polypeptide chain can affect the properties of another sub-unit in the protein.
Name two factors that can influence the interaction of R-groups.
pH
Temperature.
How can temperature affect R-group interactions?
An increase in temperature means the kinetic energy of a protein will increase, placing stress on the bonds and breaking them.
How can pH affect R-group interactions?
Changes in pH affect the concentrations of H+ and OH- ions in a solution. This changes the relative charges of the protein and places stress on polar interactions such as hydrogen bonding and ionic bonding.
What does hydrophobic mean?
To repel or fail to mix with water.
What does hydrophilic mean?
To dissolve or mix with water.
What is the Fluid mosaic model?
The Fluid mosaic model is the currently accepted model of the plasma membrane structure.
What are the main components of the plasma membrane structure?
Phospholipids
Proteins
Why are hydrophobic interactions significant in membrane structure?
Hydrophobic interactions between lipid tails hold the double membrane together.
Why are hydrophilic interactions significant in membrane structure?
Hydrophilic heads of phospholipids allow the outer layers of the membrane to be surrounded by aqueous solutions.
What are integral proteins?
Integral (or intrinsic) Proteins penetrate the hydrophobic interior of the membrane. They contain a stretch of non-polar amino acids in the hydrophobic region, which holds them in place.
What are Peripheral proteins?
Peripheral (or extrinsic) Proteins are not embedded in the lipid layer but have loose associations with the surface of the membrane.