Block A Lecture 1 - From Cells to Proteins Flashcards
Why do scientists purify proteins?
As pure proteins are needed for structure analysis, sequence analysis and therapeutic preparations
(Slide 4)
Most biological sources (such as cells) contain thousands of different proteins. What properties can proteins be separated based on?
They can be separated based on differences in size, shape, surface charge, hydrophobicity or ligand affinity
(Slide 4)
What do resolution and yield refer to in regards to protein separation techniques?
Resolution refers to the ability to separate distinct components in a mixture whereas yield refers to the quantity of the target molecule recovered after a purification process, relative to the total amount originally present in the sample.
(Slide 4)
How are resolution and yield a balance in regards to protein separation techniques?
As methods can either give a high or low resolution or a high or low yield
(Slide 4)
What are 5 pieces of information you should collect about a protein before starting a protein purification?
Information about the characteristics and properties of most important impurities should be collected. This includes:
Molecular weight
Isoelectric point (the pH at which a molecule has no net electrical charge)
Degree of hydrophobicity
Presence of carbohydrates
Free sulfhydryl groups (R-S-H) (disulphide bridges)
Stability (to temp, pH, organic solvents, oxygen, heavy metals, mechanical shear etc)
Proteolytic degradation
(Slide 5)
Why is information about a protein collected before protein purification?
In order to minimise the number of purification steps needed
(Slide 5)
Why would you want to reduce the number of protein purification steps?
As yield goes down the more steps you have
(Slide 6)
What occurs during the first stage of protein purification?
The protein is extracted from the source
(Slide 7)
What are 3 common examples of methods which are used to extract a protein from its source material?
Homogenisation, sonication and freeze-thawing.
(Slide 7)
What is homogenisation?
A mechanical process that physically breaks open cells by applying shear forces, achieved by devices which grind or shear cells.
The sample (cells or tissues) is forced through narrow gaps or subjected to high-speed mechanical agitation, which disrupts the cell membranes and releases cellular contents.
(Slide 7)
What is sonication?
It is when high-frequency sound waves are used to disrupt a cell. It is used for for breaking open bacterial cells, yeast, or small-scale cell suspensions.
The waves create alternating high and low-pressure cycles in the liquid, leading to the formation of microbubbles (cavitation). When these bubbles collapse, they generate shock waves that physically disrupt cell membranes.
It’s effective to deal with tough cells.
(Slide 7)
What is freeze-thawing?
Repeatedly freezing and thawing a sample to disrupt cell membranes.
When cells are frozen, ice crystals form inside and outside the cells. The formation and expansion of these crystals mechanically disrupt cell membranes. Thawing allows the cellular contents to leak out. Repeating the cycle enhances the disruption.
This method is often used for gentle cell lysis when preserving the activity of enzymes or other labile molecules is important
(Slide 7)
Why are protease inhibitors included during protein purification, and why is the extract kept on ice?
Protease inhibitors are included prevent proteolytic degradation of the target proteins by endogenous proteases released from lysed cells.
The extract is kept on ice to also prevent protein degradation
(Slide 7)
What is crude cell lysate?
The unprocessed mixture obtained after breaking open cells to release their intracellular contents
(Slide 8)
Lipids and nucleic acids are components of crude cell lysate. What are 2 examples of ways each of these can be removed?
Lipids: Removed by centrifugation (the lipids will float)
Removal by adsorption
Nucleic Acids
Removal by precipitation
Addition of nucleases
(Slide 8)
What are 3 examples of methods which are used in the initial fractionation of clear lysate?
Ultrafiltration
Precipitation
Fractional Precipitation
(Slide 9)
What is ultrafiltration and what is it used for?
Liquid is subjected to pressure, which forces it through the membrane. The size of the pores in the membrane allow for the selective removal of substances based on their size and molecular weight
It has a cutoff limit for separation from 1 kDa to 300 kDa and is used for removal of salts and for concentration of the protein solution
(Slide 9)
Why would you want to concentrate a protein solution?
As it allows for a higher concentration of the target protein in a smaller volume
(Slide 9)
What is precipitation used for?
As a means to concentrate the solution.
Also to remove most of the bulk proteins and other contaminants, such as proteases and membrane fragments
(Slide 9)
What is fractional precipitation, and what is it used for?
A solvent or precipitating agent is added to the mixture in a controlled manner, causing different substances to precipitate out at different stages. The process works because different substances will reach their saturation point (where they can no longer stay dissolved) at different concentrations of the precipitating agent
(Slide 9)
What is fractional precipitation used for?
To precipitate bulk proteins and to remove them and residual particulate matter via centrifugation.
The protein of interest is then precipitated afterwards from the supernatant or the protein is purified from the supernatant by column chromatography
(Slide 9)
Why are most proteins soluble?
As there are charged groups on their surface which are solvated by water
(Slide 10)
What can happen if a protein’s ability to interact with water decreases?
It can lead to decreased solubility and precipitation due to protein aggregation.
(Slide 10)
If a protein’s ability to interact with water decreases, it can lead to decreased solubility and precipitation due to protein aggregation. What are 4 examples of how this can happen?
Answers Include:
Anti-chaotropic salts - increase the hydrophobic effect in solution, as salt competes with the protein for the interaction with water.
Organic solvents - they replace water, which decreases solubility
Organic polymers
Changing the pH - the lowest solubility is at the isoelectric point
Changing the temperature
(Slide 10)
What is chromatography?
A laboratory technique used to separate mixtures into their individual components. It relies on the differential affinities of substances in a mixture for two phases: a stationary phase (solid or liquid) and a mobile phase (liquid or gas)
The separation occurs because different substances move through or interact with these phases at different rates
(Slide 14)
What is the mobile phase and stationary phase in chromatography?
The mobile phase is the solution / solvent whereas the stationary phase is solid resin
(Slide 14)
What is the setup in chromatography?
The resin is usually packed into a column and the mobile phase is passed through it.
The properties of the protein then determine how well it binds to the resin, enabling separation
(Slide 14)
What is column chromatography?
A specific type of chromatography where a sample is separated as it moves through a column that contains a stationary phase.
The sample mixture is applied at the top of the column, and a mobile phase (usually a liquid) is passed through the column, causing the different components to separate based on their differing affinities for the stationary phase
(Slide 16)
What is the set-up in column chromatography?
It’s usually an upright column made of plastic, glass or stainless steel filled with column material (a chromatographic medium or adsorption medium)
(Slide 16)
What does “minus-column” mean in the context of column chroatography?
When the column retains most of the proteins, but not the wanted protein
(Slide 16)
What is a “plus-column” in the context of column chromatography?
The column retrains the wanted protein, but most of the other proteins up in the flow through
(Slide 16)
What is an analyte in column chromatography?
The substance which is to be analysed.
(Slide 17)
What is the eluate in column chromatography?
The mobile phase leaving the column, which removes the sample components
(Slide 18)
What is flow rate in column chromatography?
The volume of fluid which passes through a given surface per unit time
(Slide 18)
What is “bed volume” in column chroamatography?
Another term for column volume for a packed column
(Slide 18)
What is “bed height” in column chroamatography?
The heigh of the column material in a packed column
(Slide 18)
What is retention time (tR) in column chromatography?
The time it takes for a particular analyte to pass through the system
(Slide 19)
What is retention volume (VR) in column chroamatography?
The volume which is required for a substance to undergo elution
(Slide 19)
What is “exclusion volume” (V0) in column chroamatography?
The volume on the mobile phase on the column
(Slide 19)
What is isocratic separation?
The composition of the solvent in the mobile phase which remains constant during the separation
(Slide 19)
What is gradient separation?
The composition of the solvent which changes stepwise or continuously during elusion
(Slide 19)
What are 4 key properties of the material used in the stationary phase of chromatography?
Answers include:
Neutral + hydrophilic
Inert (no protein binding)
It’s functional groups generate different protein-binding properties
It has chemical and physical stability
Various, but constant pore sizes
(Slide 22)
What are 3 ways which fractionation can be detected?
Measuring biological activity, using enzymatic assays with 1 unit being equal to 1 µmol of substrate being converted a minute
Use of antibodies in immunoblotting
Measurement of the protein content
(Slides 24 and 25)
What are 2 examples of way which protein content can be measured?
UV absorption during elution
Staining methods which can be used to detect peptide bonds or aromatic side chains
(Slide 25)
How does UV absorption during elution work?
Proteins absorb UV light at specific wavelengths due to their chemical structure. E.g 280 nm = absorption by tryptophan and tyrosine, 205 nm = absorption by peptide bonds
(Slide 25)
What are 3 examples of staining methods which can be used to detect peptide bonds or aromatic side chains?
Answers Include:
Biuret-assay - detection of peptide bonds and tyrosine residues (540 nm)
Lowry-Folin-Ciocalteau - detecting of peptide bonds, tyrosine, tryptophan, cysteine, cystin and histidine residues (absorption at 750, 650 or 540 nm)
Bicinchonin acid-assay (BCA-assay) - detects peptide bonds and tyrosine, tryptophan, cysteine and cystin residues (absorption at 562 nm)
Bradford-assay - detects cationic and non-polar hydrophobic side chains (absorption at 595 nm)
(Slide 25)
What are 5 examples of column chromatography methods?
Answers include:
Gel filtration
Ion exchange chromatography
Chromatofocussing
Hydrophobic interaction chromatography
Reversed-phase chromatography
Affinity chromatography
Affinity chromatography via immobilised metal ions (IMAC)
Covalent chromatography
(Slide 26)
What are 6 protein features which can be used to separate them for purification?
Charged groups (such as asp, glu, lys) (used in ion exchange chromatography and chromatofocusing)
Metal chelating groups (His, trp, cus - used in metal chelate chromatography)
Binding site (used in biospecific affinity chromatography)
Hydrophobic patches (phe, trp, ile, leu, val - used in hydrophobic interaction chromatography)
Thiol groups (-SH groups, used in covalent chromatography)
Size and shape (used in gel chromatography)
(Slide 27)
