AH Biology

what can present a hazard in a lab

substances, organisms and equipment

what do the hazards in the lab include

toxic and corrosive chemicals, heat and flammable substances, pathogenic organisms, mechanical equipment

what is risk defined as

the likelihood of harm arising from exposure to a hazard

what does risk assessment involve

identifying possible risks and the control measures to minimise them

what do control measures include

Using appropriate handling techniques, protective clothing and equipment and aseptic techniques

What are aseptic techniques

procedures in place to prevent contamination this includes sterilising equipment and workplaces

what is a linear dilution series

A range of dilutions that differ by an equal interval, for example - 0.1M, 0.2M, 0.3M...

what is a log solutions series

a range of dilutions that differ by a constant proportion, for example - 10^-1, 10^-2, 10^-3...

how is a standard curve produced

by plotting measured values for known concentrations

what is a standard curve used for

to determine the concentration of an unknown solution

what are buffers used for

to control pH; the addition of acid or alkali only has a small effect on its (buffer) pH, allowing the pH of a reaction mixture to be kept constant.

why is a colorimeter used

to quantify the concentration and turbidity of a solution

what is turbidity

the measure of relative clarity of a liquid

how do you use a colorimeter

the colorimeter is calibrated using an appropriate blank as a baseline. the measurement of absorbance is used to determine the concentration of a coloured solution using a suitable wavelength filter. the measurement of percentage transmission is used to determine turbidity.

what is centrifugation

it's a technique used to separate substances of differing density. more dense components settle in a pellet and less-dense components remain in the supernatant.

what can paper and thin layer chromatography be used for

for separating different substances such as amino acids and sugar

what does the speed each solute travels on the chromatogram depend on

its solubility in the solvent used

What is affinity chromatography

it's a separation technique in which soluble target proteins with a high affinity in a mixture become attached to specific molecules as the mixture passes down a column. non-target molecules with a weaker affinity are washed out.

what is gel electrophoresis

it's a technique that can be used to separate proteins and nucleic acids

what happens in gel electrophoresis

charged macromolecules move through an electric field applied to a gel matrix

what does native gel electrophoresis do

it separates proteins and nucleic acids by their shape, size and charge

why does native gel electrophoresis separate by size, shape and charge

because native gels don't denature molecules

why does SDS-PAGE separate proteins by size alone

because it gives all the molecules an equally negative charge and denatures them

how can proteins be separated from a mixture

using their isoelectric points (IEPs)

what is an IEP

the pH at which a soluble protein has no net charge will precipitate out of solution

what happens if a solution is buffered to a specific pH

only the proteins that have an IEP of that pH will precipitate

How can soluble proteins be separated

using an electric field and a pH gradient

what happens when a protein has no net charge

it stops migrating though the gel at it's IEP in the pH gradient

how can proteins be detected

using antibodies

what are immunoassay techniques used for

To detect and identify specific proteins. these techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies

What are monoclonal antibodies

Stocks of identical antibodies that are specific to a particular antigen

what is an antibody specific to the protein antigen linked to

a chemical 'label'

what is a chemical 'label'

often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used

what is western blotting

it's a technique used after SDS-PAGE electrophoresis. in western blotting, the separated proteins from the gel are transferred (blotted) onto a solid medium

what do specific antibodies need to identify proteins

reporter enzymes attached to the antibodies

what is bright-field microscopy used for

to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells

why does fluorescence microscopy use specific fluorescent labels

to bind to and visualise certain molecules or structures within cells or tissues

what do aseptic techniques eliminate when culturing microorganisms or cells

they eliminate unwanted microbial contaminants

how do aseptic techniques sterilise equipment and culture media

by heat or chemical means, which then means the subsequent exclusion of microbial contaminants

how can a microbial culture be started

by using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients

how are animal cells grown

in a medium containing growth factors from serum

what are growth factors

they are proteins that promote cell growth and proliferation

what are growth factors essential for

the culture of most animal cells

difference between primary cell lines and tumour cell lines in a culture

primary cell lines can only divide a limited amount of times whereas tumour cell lines can perform unlimited divisions

What does plating out of a liquid microbial culture on solid media allow

the number of colony-forming units to be counted and the density of cells in the culture estimated

Why is serial dilution often needed

to achieve a suitable colony count

what is used to estimate cell numbers in a liquid cell culture

haemocytometers

what is a haemocytometer

microscopic grid used to estimate the total number of cells within a cell culture

what is vital staining required for

to identify and count viable cells

Define proteome

The entire set of proteins expressed by a genome

Is the proteome greater or smaller than the number of genes in an organism? Why?

The proteome is larger than the number of genes, particularly in eukaryotes, because more than one protein can be produced from a single gene as a result of alternative RNA splicing, in which introns are removed from RNA transcripts and exons are retained

Are all genes expressed as proteins

No, not all genes are expressed as proteins in a particular cell type

What are genes that do not code for proteins called

Non-coding RNA genes

Give examples of things non-coding RNA genes code for

tRNA, rRNA and RNA molecules that control the expression of other genes

Is the set of proteins expressed by a given cell fixed

No, the set of proteins expressed by a given cell type can vary over time and under different conditions

Give examples of factors which affect the set of proteins expressed by a given cell

The metabolic activity of a cell, cellular stress, the response to signalling molecules and diseased versus healthy cells

Describe intracellular membranes in eukaryotic cells

Eukaryotic cells have a system of internal membranes which increases the total area of the membrane

Why do eukaryotic cells have internal membranes

Because of their size, eukaryotes have a relatively small surface area to volume ratio. The plasma membrane of eukaryotic cells is therefore too small an area to carry out all the vital functions carried out by membranes

Describe the structure of the endoplasmic reticulum (ER)

The endoplasmic reticulum (ER) forms a network of membrane tubules continuous with the nuclear membrane

Describe the structure of the Golgi apparatus

The Golgi apparatus is a series of flattened membrane discs

Describe the structure/role of lysosomes

Lysosomes are membrane-bound organelles containing a variety of hydrolases that digest proteins, lipids, nucleic acids and carbohydrates

Describe the role of vesicles

Vesicles transport materials between membrane compartments

What is synthesised in the ER

the components of membranes, phospholipids and proteins

What are phospholipids

a type of lipid that form the main component of the cell membrane

Where are lipids and proteins synthesised?

endoplasmic reticulum

Describe the difference between the Rough ER (RER) and smooth ER (SER)

Rough ER has ribosomes on its cystolic face while smooth ER lacks ribosomes

Where are lipids produced

Smooth ER (and then inserted into its membrane)

Where does the synthesis of all proteins begin

cytosolic ribosomes

Where are cytosolic proteins produced

cytosolic ribosomes. these proteins remain in the cytosol

Describe the location of transmembrane protein production.

Synthesis begins in the cytosolic ribosomes but is completed when the relevant cytosolic ribosomes dock with the ER forming RER.

What is a signal sequence

A short stretch of amino acids at one end of a polypeptide that determines the eventual location of a protein in a cell

Describe the role of the signal sequence

Transmembrane proteins carry a signal sequence, which halts translation and directs the ribosome synthesising the protein to dock with the ER, forming RER.

What happens to proteins once they are in the ER

Translation continues after docking and the protein is inserted into the membrane of the ER. They are then transported by vesicles that bud off from the ER and fuse with the Golgi apparatus

What happens when proteins move through the Golgi apparatus

they undergo post- translational modification and the addition of carbohydrate groups is the major modification.

How do molecules move through the Golgi apparatus

Molecules move through the Golgi discs in vesicles that bud off from one disc and fuse to the next one in the stack.

How are glycoproteins formed

Within the Golgi apparatus, enzymes catalyse the addition of various sugars (carbohydrates) in multiple steps to form glycoproteins

What is formed from post-translational modification in the Golgi apparatus

glycoproteins

What is the function of vesicles that leave the Golgi apparatus

They take transmembrane proteins to the plasma membrane and lysosomes.

Describe the movement of vesicles

Vesicles move along microtubules to other membranes and fuse with them within the cell

Give the location of translation of proteins which are secreted from the cell.

Proteins for secretion are translated in ribosomes on the RER and enter its lumen

Give examples of proteins for secretion

Peptide hormones and digestive enzymes

Describe the secretory pathway

Proteins for secretion are translated in ribosomes on the RER and enter its lumen. The proteins move through the Golgi apparatus and are then packaged into secretory vesicles. Secretory vesicles move to, and fuse with, the plasma membrane, releasing the proteins out of the cell.

What are many secreted proteins synthesised as

inactive precursors which require proteolytic cleavage to produce active proteins

What is proteolytic cleavage

another type of post-translational modification

Give another example of post-translational modification (not addition of a carbohydrate group)

proteolytic cleavage

What is an example of secreted proteins that require proteolytic cleavage to become active

Digestive enzymes are one example of secreted proteins that require proteolytic cleavage of inactive precursors to become active.

What does the amino acid sequence determine

protein structure

What are proteins

polymers of amino acid monomers

Name the bonds which link amino acids together in proteins

Amino acids are linked by peptide bonds to form polypeptides

What is the structure of a peptide bond

Describe the basic structure of an amino acid

Amino acids have the same basic structure, differing only in the R group present

Describe how R groups vary

R groups of amino acids vary in size, shape, charge, hydrogen bonding capacity and chemical reactivity

How are amino acids classified

Amino acids are classified according to their R groups: basic (positively charged), acidic (negatively charged), polar or hydrophobic

How can hydrophobic R groups be identified

By their hydrocarbon group. They are non-polar

How can polar R groups be identified

By their hydrophilic groups such as carbonyl (C\=O), hydroxyl (OH) or amine (NH) groups

How can acidic R groups be identified

By their carboxylic acid group (COOH/COO-) and they are negatively charged.

How can basic R groups be identified

By their amine group. They are positively charged and hydrophilic

What does the wide range of protein function result from

The diversity of R groups

What is the primary structure of a protein

the sequence in which the amino acids are synthesised into the polypeptide

Describe the secondary structure of a protein

Hydrogen bonding along the backbone of the protein strand results in regions of secondary structure - alpha helices, parallel or anti-parallel beta-pleated sheets, or turns