Unit 1 Flashcards
Dilution
- Reducing the concentration of a substance in a solution
1. Serial (log) dilution
2. Linear dilution
Serial dilution
Each dilution acts as the stock to make the next dilution in the series
Each concentration depends on those made before and any earlier measurement errors are compounded in later dilutions.
Each step is a ten fold dilution.
Linear Dilution
Different volumes of stock solution are added to different volumes of solvent.
Each concentration is made individually so any measurement errors affect only one concentration.
Colorimeter
Used to measure the concentration of a pigment in a solution -> does this by measuring the absorbance of specific wavelengths of light by a solution
How does a colorimeter work?
A beam of light (at a specific wavelength) is passed through a cuvette containing a sample solution. Some of the light is absorbed by the sample; therefore a lower intensity hits the detector and the machine will display an absorbance value.
Machine is calibrated using a cuvette with distilled water. This acts as a control as it provides a reading with 0 absorbance.
pH of solution
pH is a logarithmic measure of hydrogen ion concentration.
pH can be measured using an electronic pH meter or a pH indicator.
pH buffer
A solution whose pH changes very little when a small amount of acid or base is added to it.
Used as a means of keeping pH at a nearly constant value.
Ways of determining an unknown concentration
Standard curve or titration
Standard curve
A standard curve is made by plotting the absorbance readings of a series of known concentrations.
The graph can be used as a reference for any samples of unknown concentrations of the same substance or culture.
Titration
A burette delivers a solution of known volume and concentration to a solution of unknown concentration.
Separation techniques
Centrifugation.
Protein electrophoresis.
Iso-electric point.
Paper, thin layer and affinity chromatography.
Substances can be separated according to their solubility, size, shape or charge.
Centrifugation
A centrifuge is a piece of equipment that can spin a sample at very high speeds.
Substances are separated according to their size and density.
- the largest and densest materials separate out first and form a pellet at the bottom of the tube
- the liquid that remains above the pellet is called the supernatant
Ensure the tubes are balance.
Chromatography
Refers to a set of techniques which separate the components of a mixture
Paper and thin layer chromatography
Paper stationary phase - chromatograph paper
Thin layer chromatography stationary phase - silica gel or cellulose
Separates a mixture as components travel at different rates depending on their properties: polarity and solubility
E.g. paper contains cellulose fibres which are polar in nature; any components of the mixture which are polar will bond with the cellulose fibres relatively quickly and do not travel far up the paper
Rf value
The ratio of the distance moved by the solute from the origin and the distance moved by the solvent from the origin.
Rf = distance travelled by compound/distance travelled by solvent front
Affinity chromatography
Relies on the binding interactions between a protein and ligand.
A ligand or antibody specific for binding with the protein in question is immobilised in an agarose gel packed into a column. The mixture of proteins is poured though the column. Only the protein of interest bonds to the antibody if ligand within the column. Other proteins will not bind and can be washed away. Washing the column with a buffer of different pH, the target protein can be recovered.
Protein electrophoresis
Process separates proteins based on their size and charge.
Used current flowing through a buffer to separate proteins. The cell acts as a sieve.
Folded:
- native non denatured protein
- migration of protein through the gel depends on charge and size.
Unfolded:
- non-native denatured protein
- denature first the protein results in unfolded linear protein with uniform charge
- migration depends on size
- small proteins travel further
Iso-electric point
The isoelectic point of a protein is the pH at which it has an overall neutral charge.
Positive charge pH below isoelectric point
Negative charge pH above isoelectric point
At the isoelectric point the overall neutral charge allows the protein to form a solid and precipitate out of solution.
By changing the pH of solution, successive proteins can be separated.
Iso electric focussing
pH gradient is set up along a tube of polyacrylamide gel using a mixture of special buffers.
Each protein loaded onto the gel will move until it reaches the pH corresponding to its isoelectric point.
At this pH the protein will move no further and form a bond which can be visualised after staining.
Antibody
Y-shaped globular proteins produced by beta lymphocytes as part of the immune response of a vertebrate
Antibodies bind to specific antigens and flag them for destruction
Immunoassay
When antibodies are used to detect both the presence and concentration of a protein within solution.
Relies on the specificity of antibodies as they only recognise and bind with one antigen.
Immunoassay - how it can be identified
Antibody used must be linked to a detectable label so it can detect when binding has occurred.
Labels can be in the form of reporter enzymes which causes a colour change in the presence of a specific antigen.
ELISA (enzyme linked imunosorbent assay)
An analytical technique that uses antibodies and a colour change to identify a substance (antigen) in a solution.
A colour producing substance is added to the well and a colour is allowed to develop. Any wells which remain colourless indicate protein of interest is not in the sample. Any wells which show colour indicate a positive result so protein of interest is in sample.
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
Direct ELISA
The antigen is allowed to bind to the surface of a multiwell plate, a primary antibody, linked to a reported enzyme, is added to the well and binds to the antigen
Indirect ELISA
The antigen is allowed to bind to the surface of a multiwell plate. A primary antibody is added to the well and allowed to bind to the antigen. A secondary antibody, linked to a reporter protein is then added which binds to the primary antibody.
Sandwich ELISA
A capture antibody is bound to the surface of a multiwell plate. The antigen is added and allowed to bind to the capture antibody. A primary antibody, which binds to the antigen, is added to the well. A secondary antibody, linked to a reporter enzyme is then added, which binds to the primary antibody.
What is ELISA used for
Detection of HIV
Detection of food allergens
Screening of certain types of drugs
Fluorescent labelled protein blotting
Technique for identifying specific proteins that have been separated using gel electrophoresis.
Proteins are transferred to a membrane.
A specific antibody that is linked to a detectable label (a fluorescent tag) is added to the membrane, these will bind to their target proteins and the excess is washed away
Allows the location of the target protein to be precisely identified.
Immunohistochemistry
When antibodies are used to detect the presence of a particular antigen within a tissue.
Advantage is that it is capable of showing exactly where a certain protein is being expressed within a tissue sample.
Monoclonal antibodies
A supply of antibodies that are identical and will bind to exactly the same feature of the antigen
Formation of monoclonal antibodies
Hybridomas are formed by fusion of a Beta lymphocyte (produced by antibodies) with a myeloma (cancer) cells from an immortal cell line, using polyethylene glycol (PEG)
Beta lymphocytes do not divide in culture and so are fused with myeloma
Bright field microscopy
A sample is mounted on a slide and illuminated below.
Light is transmitted through the specimen to the objective lens and then to the eyepiece where the image can be observed.
The image of the sample that is produced is usually darker than the background which appears bright.
Samples are often stained before being viewed.
Fluorescence microscopy
Specific protein structures have fluorescent markers added to them.
The cell can be placed on a slide and the protein structure can be visualised using a fluorescence microscope.
Fluorescent molecule
Absorbs a specific wavelength of light then emits a different (longer) wavelength.
Absorbs light of one colour and emits light of another colour.
Immunofluorescence
When antibodies are used to fluorescently tag protein structures.
A primary antibody which is specific to the protein being visualised is introduced to a cell sample.
A secondary antibody attached to a fluorescent tag is then added which binds to the primary antibody.
Haemocytometer
Graduated microscope slide used to count cell density.
Can be used to make total cell counts or if vital staining is used to distinguish living cells, it can be used to make viable cell counts.
Cell culture
- what is it
- what does it produce
- what environmental factors must be controlled
- what does a typical culture medium contain
The ability to grow cells in an artificial laboratory environment.
Produces many genetically identical clones of an initial cell sample.
Optimum conditions are provided in terms of nutrients, pH, temperature and gases which must be controlled.
A typical culture medium contains water, salts, amino acids, vitamins and glucose
what does animal cell culture require
Requires media containing growth factors for serum
e.g. bovine serum which contains growth factors that promote cell proliferation.
What is an inoculum
The cells that are used to inoculate culture media
Growing cells are added in a volume of liquid medium from a previous culture
What are proteolytic enzymes
Small pieces of tissue
Aseptic techniques
Precautions taken to prevent contamination; the use of sterile materials and the appropriate treatment of the source tissue are vital to prevent accidental inoculation with unwanted cells or spores.
- wiping down surfaces, containers and dishes with ethanol
- hands should be washed
- protective equipment
Genome
An organisms genome is its complete set of DNA including both the protein coding genes and the non-coding regions of the DNA
Proteome
The entire set of proteins that can be expressed from a genome.
Much larger than the number of genes
Alternative RNA splicing
Depending on which RNA segments are treated as expand and introns in a primary RNA transcript of a gene, different mature mRNA molecules are produced
Post translational modification
The polypeptide made at the ribosome can be cut and combined in different ways and can also have phosphate or carbohydrate groups added to it
Polypeptides
Polymers of amino acid monomers
What happens when polypeptides undergo post translational modification?
The polymers become proteins.
What happens when an amino acid is in an aqueous solution?
Basic - The NH2 group will gain a hydrogen to form NH3+
Acidic - The COOH group will lose a hydrogen to form COO-
Polar - The -OH is slightly charged so can form hydrogen bonds with other molecules such as water.
Hydrophobic - Hydrocarbon groups carry no charge so do not form any hydrogen bonds with water so they do not mix readily with water
What functional group does an acidic (negatively charged) amino acid have?
-COOH
What functional group does a basic (positively charged) amino acid have?
-NH2
What functional group does a polar amino acid have?
-OH
What functional group does a hydrophobic amino acid have?
Hydrocarbon
How are amino acids linked together?
Linked together during translation at the ribosome.
An enzyme causes a condensation reaction between 2 adjacent amino acids. A water molecule is removed by joining the OH of the COOH of one amino acid to a hydrogen from the NH2 of the other amino acid.
What bond links amino acids together
Peptide bonds
What determines a proteins structure and function?
The amino acid sequence
What is the primary structure
The sequence in which the amino acids are synthesised into the polypeptide.
The R groups are usually far apart
What is the secondary structure?
The secondary structure is stabilised by hydrogen bonds along the backbone of the polypeptide strand.
These hydrogen bonds exist between different peptide bonds in the chain.
The hydrogen of the N-H has a weak positive charge so it is electrically attracted to the weak negative charge on the oxygen of the C=O of another peptide bond.
The polypeptide chain can form turns where the chain folds back on itself. The folding in the secondary structure brings R groups close together to interact.
Alpha helix secondary structure
A spiral with the R groups sticking outwards.
Beta sheet secondary structure
The beta sheet has parts of the polypeptide chain running alongside each other to form a corrugated sheet, with the R groups sitting above and below.
The beta sheets are either antiparallel (chains in the opposite directions with respect to N-C polarity)
Or they are parallel (chains in the same direction with respect to N-C polarity)
What is the tertiary structure
The final folded shape of the polypeptide.
How is the 3D conformation containing regions of secondary structure in the tertiary structure stabilised?
Stabilised in position by interactions between R groups of amino acids
Hydrophobic interactions between R groups
As a polypeptide folds into its functional shape, the hydrophobic R groups are repelled by water and so usually end up to the inside of the polypeptide.
Ionic bond interactions between R groups
COOH and NH2 groups ionise to become COO- and NH3+. These groups are strongly charged so attract each other.
van der Waals interactions between R groups
Very weak attractions between the electron clouds of atoms.
Hydrogen bonds are a particularly strong example of these.
Disulphide bridges between R groups
Covalent bonds form between sulfur containing R groups of cysteines
What happens when the tertiary structure experiences higher temperatures
The structure becomes destabilised leading to denaturation. The increased heat provides more kinetic energy so the polypeptide chain shakes more, breaking the weaker ionic bonds, van der Waals interactions and hydrogen bonds.
What happens to tertiary structures when pH changes
The changes in pH affect the ionisation if the acidic and basic R groups, changing the charge they carry so they no longer bond correctly and the polypeptide unfolds
What happens when a protein has more than one polypeptide subunit
It forms the quaternary structure when the subunits connect by bonding between their R groups.
There is cooperativity between their polypeptide subunits. The bonding between them means that changes to the shape of one subunit can affect properties of other subunits
What is a prosthetic group
A non-protein group which is strongly bound to a polypeptide unit and it is essential for the proteins function.
What is the position of the R groups determined by?
The primary structure
How do R groups determine the structure of a protein?
The primary structure means that the R groups have specific locations in the polypeptide chain. This determines:
- the formation of regions of secondary structure in the chain
- the interactions that can occur between R groups in the tertiary structure
- the interactions with other polypeptide chains in the quaternary structure
- the interactions with prosthetic groups
What is a ligand?
A molecule that can bind to a protein
How do R groups allow the binding of ligands?
The R groups that are not involved in protein folding will be on the outer surface of the protein, so they are available for ligand binding. This is important because this binding changes the conformation of the protein which changes its function or activity.
How do R groups at the surface of a protein determine it’s location within a cell?
The balance of hydrophobic R groups and hydrophilic R groups on the proteins surface will influence the solubility or the protein in the aqueous cytoplasm or interact with the hydrophobic layers of membranes
Important roles of DNA
The packaging is achieved by wrapping the DNA in tight coils around histone proteins to form bead like structures called nucleosomes, packing the DNA into chromosomes
DNA as a ligand
The sugar phosphate backbone of DNA is negatively charged which allows it to bind to the positively charged K groups arranged to the outside of the histone protein
Complementary chemistry (ligands and binding)
The binding site has charged polar and non polar R groups arranged in a way that matches the charged polar and non polar areas on the ligand
Membrane proteins
The cell membrane is embedded with proteins which form a patchy mosaic:
- integral proteins
- peripheral proteins
Integral proteins
Penetrate the hydrophobic interior of the phospholipid bilayer
Held in place by strong hydrophobic interactions with the phospholipid tails
R groups (hydrophobic) allow strong interactions
Transmembrane protein span width of the membrane
Peripheral proteins
Form weak bonds (with heads of phospholipids or exposed parts of integral proteins) to the surface of the membrane and are not embedded in the bilayer.
Have few k groups interacting with phospholipids
What can and can’t pass through the phospholipid bilayer
Non polar molecules can pass through the hydrophobic region of the phospholipid bilayer
Charged ions and most polar molecules can only cross the membrane through proteins suspended in the phospholipid bilayer
Phospholipids
Hydrophobic heads - repelled by water
Hydrophilic heads - attracted by water
The phospholipids are constantly changing position which gives the membrane its fluid quality
Fluid mosaic model of membrane structure
The phospholipid bilayer can move so it is fluid and the separate proteins are like small pieces of a mosaic
Coding and non coding sequences
Coding sequences - exons
Non coding - introns
Introns aswell as exons are transcribed into RNA
In RNA splicing the introns are removed and exons are joined together
When RNA is spliced it is a functional mRNA molecule
Induced fit of a protein
The conformational change is called induced fir and it helps to further increase the binding and interaction between the active site and the substrate
What is activation energy
The energy needed to allow a reaction to occur.
The activation energy of a reaction is lowered by the stressing of the bonds in the substrate
How is the rate of product formation by a metabolic pathway regulated?
By raising or lowering the activity of just one enzyme in the pathway
Allosteric enzymes
Enzymes that have their activity regulated by altering their conformation
What causes the change in conformation of an allosteric enzyme?
A modulator binds to the enzyme at a secondary binding site (also called an allosteric enzyme).
Positive modulators increase the affinity and so increase enzyme activity.
Negative modulators decrease the affinity and the activity
What does the change in enzyme shape do?
Alters the affinity of the active site for its substrate which moderates the effectiveness of the substrate binding and the enzyme activity
