Advanced Higher Biology - Unit 1 - Cells and proteins

What is a hazard?

A hazard is any agent (chemical, biological, physical) that has the potential to cause harm

What is a risk?

The risk is the likelihood of harm arising from exposure to a hazard.

What is a risk assessment?

A risk assessment identified hazards, assesses risk levels, and proposes control measures.

What are hazards in the biology lab?

Toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms and mechanical equipment.

Explain what the difference is between a hazard and a risk.

A hazard is anything with tye potential to cause harm (e.g. a bottle of ethanol - it is flammable). Risk is the likelihood that harm will actually occur from exposure to that hazard (e.g. the risk of fire of ethanol is used near a naked flame).

What is a linear dilution series?

Linear dilution series differ by an equal interval, for example 0.1, 0.2, 0.3.

What is a log dilution series?

Log dilution series differ by a constant proportion, for example 10-1, 10-2, 10-3.

What can colorimeters be used for?

Quantify concentration and turbidity. Absorbance is used to determine the concentration of a coloured solution using suitable wavelength filters. Percentage transmission is used to determine turbidity, such as cells in suspension.

What is a standard curve?

A standard curve is produced by plotting absorbance values for known concentrations so that unknown concentrations can be determined.

What is the Bradford test?

A test which allows the concentration of protein in a solution to be determined.

How does the Bradford's test work?

Produce a standard curve by adding a set volume of Bradford reagent to a set volume of different known concentrations of protein being tested. The absorbance at 595 nm is measured for each concentration and a graph is plotted to produce the protein standard curve.

Explain why a blank is used when setting up the colorimeter?

The blanks account for any absorbance due to the solvent or reagents themselves.

What is a buffer?

A solution that resists changes in pH when small amounts of acid or alkali are added.

When a cell extract is centrifuged at low speed. Which components would you expect to find in the pellet?

Larger, denser components such as unbroken cells, cell debris and nuclei, starch, proteins.

When a cell extract is centrifuged at low speed. Which components would you expect to find in the supernatent?

Smaller, less dense components such as mitochondria, ribosomes and soluble proteins.

What does the distance that the solute travels along the chromatogram depend on?

It's solubility in the solvent used.

What does TLC chromatography use?

A stationary phase of silica gel on a glass plate, giving faster runs and better separation than paper.

State two advantages of TLC over paper chromatography.

It gives faster separation (shorter run times) and gives better resolution/separation between substances due to finer, more uniform silica gel stationary phase.

What is affinity chromatography used for?

To isolate a specific protein from a mixture based on its specific binding affinity.

Describe affinity chromatography.

A solid matrix or gel column is created with specific molecules (often antibodies or ligands) bound to it. Soluble target proteins, with a high affinity for these molecules, bind to the column as the mixture passes through. Non-target molecules with weaker affinity are washed out. The bound protein is elite from the column by washing with a buffer.

What does gel electrophoresis do?

Separates charges macromolecules (proteins and nuclei acids) by size, shape and/or charge using an electric field.

How does gel electrophoresis work?

Charged macromolecules move through electric field applied to a gel matrix. Proteins travel through the electrode with an opposite charge. Smaller proteins travel faster through the gel than larger proteins.

What does native gel electrophoresis do?

Separates proteins by their size, shape and charge.

What does SDS-PAGE gel electrophoresis do?

Separates proteins by their size alone and proteins are denatured. It gives all the molecules an equally negative charge, proteins migrate to the positive electrode. Small proteins travel further through the gel than larger proteins.

What is the use of SDS-PAGE?

Determining relative molecular mass of proteins.

A researcher uses SDS-PAGE to separate five proteins. After staining, four bands appear on the gel. What does this tell you about the proteins?

Two of the original proteins must be the same size (or very similar molecular mass), as they have co-migrated into a single band. Since SDS-PAGE separates by size only, it cannot distinguish proteins of the same molecular mass.

What is the isoelectric point?

The specific pH at which a soluble protein has no net charge and will precipitate out of solution.

What will happen if a solution is buffered to a specific pH?

Only proteins with an IEP equal to that pH will precipitate.

What happens in isoelectric focusing?

Proteins are placed in a gel with a IEP gradient. Each protein migrates until it reaches its IEP. A protein stops migrating through the gel at its IEP because it has no net charge to move in the electric field.

Protein A has an IEP of pH 5. Protein B has an IEP of pH 8. A mixture of both proteins is placed in a buffer at pH 5. What would you observe?

Protein A would precipitate out of solution (it is at its IEP so has no net charge). Proteins B would remain in solution (the pH is below its IEP of 8, so it carries a net positive charge). The result is separation of the two proteins.

Explain how isoelectric focusing can be used to separate proteins from a mixture.

Isoelectric focusing uses a gel containing a pH gradient. When an electric field as applied, each protein migrates through the gel until it reaches the pH region where it has no net charge and so will precipitate out of solution and stop moving. This is its isoelectric point. Proteins with different IEP's stop at different positions in the gel and are therefore separated. After staining, distinct bands are visible.

What do immunoassay techniques use?

Use antibodies to detect and identify specific proteins.

Describe immunoassays.

Immunoassays rely on the ability of an antibody to recognise and bind a specific molecules in a complex mixture. The molecule bound by an antibody is referred to as an antigen. These techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies. An antibody specific to the target is linked to a chemical label often a reporter enzyme that produces a colour change. Other labels include chemiluminescence and fluorescence.

What is Elisa used for?

To detect the presence of specific antigens or antibodies in a sample. There are three types: direct Elisa, indirect Elisa and competitive Elisa.

What happens during direct Elisa?

The antigen is allowed to bind to a multi well plate. A primary antibody linked to a reporter enzyme is added.

What happens during indirect Elisa?

A primary antibody binds to the antigen. A secondary antibody, linked to a reporter enzyme, then binds to the primary antibody.

What happens during competitor Elisa?

A capture antibody is first bound to the plate. The antigen binds to the capture antibody, then primary and secondary antibodies are added.

What does the colour of the wells in Elisa indicate?

Colourless - negative and colour change - positive.

State two uses of Elisa in medicine or research.

- Detecting HIV antibodies in blood samples.

- Testing for allergens.

In an indirect Elisa, why is a secondary antibody used rather than linking the reporter enzyme directly to the primary antibody?

Increases sensitivity : using a secondary antibody (which binds to the primary antibody, increases the signal because multiple secondary antibodies can bind to one primary antibody. So,more reporter enzyme is present or antigen molecule, increasing sensitivity since more reporter enzyme molecules changing substrate to a coloured product so amplifying the colour change.

Explain what a positive Elisa result indicates and how the colour change is produced.

A positive result indicates the target antigen (or antibody) is present in the sample. The reporter enzyme attached to the antibody catalyses the conversion of a colourless substrate into a coloured product producing a visible colour change in the well. The intensity of colour is proportional to the amount of antigen present.

What is western blotting used for?

To identify specific proteins after they have been separated by SDS-PAGE.

What is western blotting?

Separated proteins are blotter (transferred) from the gel onto a solid/nitrocellulose medium (membrane). Proteins are then identified using specific antibodies with reporter enzymes attached.

Describe the full process of western blotting.

1. Proteins are extracted from cells and separated by SDS-PAGE (by size).

2. Proteins are transferred (blotted) from the gel onto a nitrocellulose membrane using an electric current.

3. The membrane is incubated with a primary antibody specific for the target protein.

4. A secondary antibody linked to a reporter enzyme is added, it binds to the primary antibody.

5. A substrate is added; the reporter enzyme converts it to a coloured product, revealing the position (and therefore size) of the target protein.

Explain the role of the reporter enzyme in Western blotting.

The reporter enzyme (e.g horseradish peroxiadse) catalyses the conversion of a colourless substrate into a coloured (or chemiluminescent) product. This produces a visible or detectable signal at the location of the target protein on the membrane.

Why must SDS-PAGE be carried out before Western blotting?

SDS-PAGE separates proteins by size so that when they are transferred to the membrane and detected by antibodies, the size of the target protein can be confirmed. Without prior separation, all proteins would be present in a single mixed spot with no size information.

What is genomics?

The study of the genome.

What is the genome?

The total genetic material in a cell: an organism's complete set of genetic instructions.

What is proteomics?

The study of the proteome.

What is the proteome?

The entire set of proteins expressed by a genome. It is larger than the number of genes, particularly in eukaryotes.

Why is the proteome larger than the genome?

Because more than one protein can be produced from a single gene as a result of alternative RNA splicing.

What is differentiation?

As a cell develops to perform its specialised function some genes are switched on and others are permanently switched off. Only the genes necessary for the specialised function of the cell are switched on, so only those proteins are produced by that cell.

What are genes that do not code for protein called?

Non-coding RNA genes

What are non-coding RNA genes transcribed to produce?

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

What is the transcriptome?

The set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced by a genome.

What are some factors affecting the set of proteins expressed by a given cell type?

The metabolic activity of the cell, cellular stress, the responses to signalling molecules and diseased vs healthy cells.

What type of membrane do eukaryotes have?

A plasma membrane.

What is a plasma membrane?

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

What system does a eukaryote have and what does it do?

A system of internal membranes (endomembrane system), which increases the total area of membrane and provides a larger surface area for vital functions to take place.

What organelles are in a eukaryote?

Golgi apparatus, Golgi vesicles, nucleolus, lysosome, microtubules, smooth endoplasmic reticulum, vacuole, centrioles, rough endoplasmic centiculum, pinocytotic vesicle.

What does the endoplasmic reticulum do?

Forms a network of membrane tubules continuous with the nuclear membrane. There are two types; rough ER and smooth ER. They are involved in membrane synthesis.

What is the difference between rough endoplasmic reticulum and smooth endoplasmic reticulum?

Rough ER has ribosomes on its cytosolic face, smooth ER lacks ribosomes.

What does the Golgi apparatus (Golgi body) do?

A series of flattened membrane discs involved in processing and packaging of proteins.

What are lysosomes?

Membrane-bound organelles containing a variety of hydrolases that digest proteins, lipids, nucleic acids and carbohydrates.

What are hydrolyses?

Enzymes that catalyse the cleavage of a covalent bond using water.

What are vesicles?

They transport materials between membrane compartments. They consist of an aqueous solution enclosed by a lipid bilayer.

What is the endoplasmic reticulum involved in?

Synthesis of both phospholipids and proteins.

What happens to lipids?

They are synthesised in the smooth endoplasmic reticulum (SER) and inserted into its membrane.

Describe the synthesis of lipids.

Precursors for lipid production are floating in the cytosol (aqueous part of the cytoplasm). Enzymes in the smooth ER membrane collect and synthesise the phospholipids and insert them into the membrane, continually expanding the ER. Sections of the membrane can then bud off and be transferred as vesicles to other parts of the internal membrane systems.

What is the cytosol?

It is the liquid found inside cells. Also known as intracellular fluid or cytoplasmic matrix.

Where does the synthesis of all proteins begin?

Cytosolic ribosomes - ribosomes in the cytoplasm not attached to the endoplasmic reticulum.

Where is the synthesis of cytosolic proteins completed?

In the cytosolic ribosomes, and these proteins remain in the cytosol.

Describe transmembrane proteins.

Transmembrane proteins carry a signal sentence, which halts translation of the protein at the cytosolic ribosome and directs the ribosome synthesising the protein to dock with the ER, forming Rough Endoplasmic Reticulum.

What is a signal sentence?

A short stretch of amino acids at one end of the polypeptide that determines the eventual location of a protein in a cell. Translation continues after docking, and the protein is inserted into the membrane of the ER.

What happens to ribosomes after the ER?

Once the proteins are made at a ribosome on the rough endoplasmic reticulum and is put into the lumen of the RER, they are transported by vesicles that bud off from the ER and fuse with the Golgi apparatus.

What happens to proteins moving through the Golgi apparatus?

The Golgi apparatus is a series of flattened membrane discs. It is involved in the transport and modification of proteins. Molecules move through the Golgi discs in vesicles that bud off from one disc and fuse to the next one in the stack. As proteins move down the Golgi apparatus they undergo post-translational modification.

Describe post-translational modification.

1. The addition of chemical groups; carbohydrate structures or phosphate groups attached to a protein can dramatically affect its charge and shape and therefore it's function. The addition of carbohydrate groups is the major modification. This is carried out by enzymes. Enzymes catalyse the addition of various sugars in multiple steps to form the carbohydrates.

2. The cleavage of a polypeptide to make an active protein. This involves cleaving (cutting) sections of the polypeptide.

What happens to proteins after the Golgi apparatus?

Vesicles that leave the Golgi apparatus take proteins to the plasma membrane and lysosomes (which has a membrane). Vesicles move along microtubles to other membranes and fuse with them from within the cell.

What are microtubles?

Structures that make up the cell's cytoskeleton and offer support and a means of transport.

What does it mean, when cells secrete substances?

They release substances made inside the cell to the outside.

What are examples of secreted proteins?

Peptide hormones, e.g. insulin and digestive enzymes.

Describe the pathway of secreted substances?

Secreted proteins are translated in ribosomes on the rough endoplasmic reticulum and enter its lumen. They bud off the RER in a vesicle and go to the Golgi apparatus. The proteins move through the Golgi apparatus and are then packaged into secretory vesicles. These secretory vesicles move to and fuse with the plasma membrane, releasing the proteins out of the cell.

What is proteolytic cleavage?

The process of breaking the peptide bonds between amino acids in proteins. It is an example of post-translational modification where the polypeptide is cut.

Describe how secretory proteins are produced in the cell.

Many secreted proteins are synthesised as inactive precursors and require proteolytic cleavage to produce active proteins.

Why are secreted proteins inactive?

An active form inside a cell would be damaging and so it is activated at the site of action.

Give a summary of the secretory pathway.

Ribosome on RER - lumen of RER - vesicle - Golgi apparatus - secretory vesicle - plasma membrane - protein released out of cell.

What are proteins?

Polymers of amino acid molymers. Amino acids are linked by peptide bonds to form polypeptides.

What is a peptide bond?

A strong covalent bond between a carbon atom of one amino acid and the nitrogen atom of another amino acid.

What are the two functional groups in an amino acid?

An amine group and an acid group.

What is the 'R' group on an amino acid?

A variable group.

What do 'R' groups of amino acids vary in?

Size, shape, charge, hydrogen bonding capacity and chemical reactivity.

What are the main categories of amino acids split into?

Acidic (negatively charged, hydrophilic), basic (positively charged, hydrophilic), polar (hydrophilic, charged), hydrophobic (non-polar).

Describe acidic R groups.

Negatively charged amino acids are hydrophilic and the key component of their R group is a Carboxylic acid group (COO-). For example - aspartic acid.

Describe a basic R group.

Positively charged amino acids are hydrophilic and the key component of their R group is an amine group. For example - Lysine.

Describe polar amino acids.

Polar amino acids are hydrophilic and the key components of their R groups are hydrophilic groups, like carbonyl (C=O), hydroxyl (OH) or amine (NH) groups or sulfydryl (SH) groups. For example - serine.

Describe non-polar hydrophobic R groups.

Amino acids with hydrophobic R-groups are non-polar and they key component of their R group is a hydrocarbon group like CH3 or an aromatic group (ring). For example - alanine.

What are the levels of protein structure?

Primary structure, secondary structure, tertiary structure and quaternary structure.

Describe primary structure.

Amino acids link by peptide bonds to form polypeptides. The primary structure is the sequence in which the amino acids are synthesised into the polypeptide.

Describe secondary structure.

Hydrogen bonding along the backbone of the protein strand results in regions of secondary structure. The secondary structure of a protein is stabilised by hydrogen bonds between atoms of the same chain. There are three types of secondary structure; alpha-helix, beta-pleated sheets, turns.

Describe alpha helix.

It is a spiral with the R groups sticking outwards.