Protein Purification

Question 1

You cloned a gene of interest with a C-terminal His tag and expressed the protein in bacteria. How would you purify the protein?

Answer 1

Affinity chromatography would be used in this case, specifically Nickel (Ni2+) immobilised onto magnetic beads as Nickel (Ni 2+ binds strongly to His-Tag

Question 2

What is Western blotting?

Answer 2

Western blotting is a laboratory technique for detecting specific proteins in a sample. It involves separating proteins by molecular weight using gel electrophoresis, transferring them to a membrane, and then using antibodies to identify and visualise the target protein.

Question 3

Why is Western blotting used?

Answer 3

The purpose of Western blotting is to confirm the presence and size of specific proteins in a sample. It is widely used in research and clinical laboratories for applications such as protein expression analysis, disease diagnosis, and validating results from other assays like ELISA.

Question 4

What is immunoblotting?

Answer 4

The process involves separating proteins based on size using gel electrophoresis, transferring them to a membrane, and then probing the membrane with antibodies that specifically bind to the target proteins. The antibodies can be visualized using various detection methods, allowing researchers to analyze the presence and abundance of proteins in biological samples.

Question 5

What is the principle of gel filtration?

Answer 5

Protein is passed through agarose gel; larger proteins pass through faster as they do not enter the pores within the gel.

Whereas large proteins can readily avoid these pores and pass-through

Question 6

When you run gel filtration on a known protein and compare its mass to the standards, and your protein does not fall within its known mass. What could be the reason for this?

Answer 6

Some proteins exist as Dimer’s and Trimer’s, so the kDa may be higher than expected.

Question 7

What is a dimer?

Answer 7

A dimer is a protein complex formed by two identical or different polypeptide chains (subunits) that are non-covalently associated or covalently linked.

Question 8

What is a Trimer?

Answer 8

A trimer is a protein complex formed by three polypeptide chains (subunits) that are associated together.

Question 9

What does SDS PAGE measure and what would a 2D measurement look at?

Answer 9

SDS PAGE determines the molecular weight of an unknown protein.

This can be done on a 2D scale by separating proteins according to their isoelectric points.

Question 10

What does “isoelectric points” mean in proteins?

Answer 10

the pH at which the protein has no net charge, making it a crucial factor in understanding protein solubility, stability, and interactions in various biochemical contexts.

Question 11

What are the advantages of using bacteria for protein purification?

Answer 11

Fast growth rate (20 min doubling time) - can generate lots of protein-expressing bacteria very quickly.

Can transform bacteria with plasmid DNA rapidly (less than 5 mins)

Relatively cheap.

Question 12

What are the disadvantages of using bacteria for protein purification?

Answer 12

Proteins may not fold correctly

High conc of protein can be insoluble (inclusion bodies)

Lack some post-translational modification (e.g. phosporylation)

Question 13

What methods of lysing bacterial cells expressing target protein? What should you do after the cells have been lysed?

Answer 13

• freeze-thawing (e.g. Liquid N2)
  -Non-ionic detergent (e.g. triton X-100)
• Sonication (Ultra-High Frequency sound)

Centrifuge again after cell lysis - supernatant contain soluble cellular material, including proteins.

Question 14

What is the principle of ammonium sulfate preciptation?

Answer 14

1.Salting Out: As ammonium sulfate is added to a protein solution, it reduces the amount of water available for protein hydration, causing some proteins to become less soluble and precipitate out of the solution.

2.Selective Precipitation: Different proteins have unique solubility profiles at various ammonium sulfate concentrations. By gradually adding ammonium sulfate, proteins can be selectively precipitated based on their solubility.

3.Centrifugation: After precipitation, the mixture is centrifuged to separate the solid precipitate (the proteins of interest) from the liquid supernatant, which contains unprecipitated proteins.

Question 15

What is ammonium sulphate precipitation?

Answer 15

Ammonium sulphate precipitation is based on the principle of differential solubility, where proteins are selectively precipitated from the solution by altering the salt concentration. By manipulating ammonium sulphate concentrations, researchers can effectively separate proteins based on their solubility characteristics, facilitating the purification of specific proteins from complex mixtures.

Question 16

how would you use differential solubility using Ammonium Sulphate precipitation to select a specific protein?

Answer 16

Different proteins have different solubilities in aqueous solutions.

By changing the amount of salt added we can solubilise specific proteins.

Question 17

Why use Ammonium Sulphate for precipitation?

Answer 17

Highly water soluble
Cheap
Available at high purity
No permanent denaturation of proteins

Question 18

What are the most common 3 methods for removing salt from your protein sample?

Answer 18

dialysis
gel filtration chromatography
diafiltration

Question 19

How does dialysis remove salt from the protein solution?

Answer 19

1. Sample is placed in a bag with semi-permeable membrane (pores)
2. Choose permeability based on target protein
3. Pores too small to allow passage of your protein but big enough to allow passage of salt ions (salt reaches Eq)
4. Several changes of buffer eventually remove the salt from your sample.

Question 20

how does gel filtration chromatography remove salt from protein solution?

Answer 20

• Load dissolved protein (and salt) onto column - flush sample through buffer
• Resin has pores that some components can enter
• Small ions enter pores of resin, whilst large proteins pass straight through.

Question 21

What does diafiltration remove salt from the protein solution?

Answer 21

1. Sample is passed through a cycle, with buffer constantly flowing.
2. At one end of the cycle a permeable filtration module is present which allows salt to pass through.
3. New buffer is constantly added in this cycle until salt is fully removed.

• Salt passes through membrane
• Protein is retained in sample.

Question 22

What is the principle of using heat denaturing to purity protein sample?

Answer 22

Heat denatures the tertiary structure of proteins exposing hydrophobic interior = aggregation

If your protein is thermis stable at a certain temp.. you can heat to this temp and either remove your proteins as ppt or soluble.

Question 23

What is the principle of using affinity chromatography to purify protein samples?

Answer 23

1. affinity matrix composed of an affinity molecule bound to a solid support (e.g. sepharose beads)
2. Affinity matrix specfically recognises protein of intrest
3. Protein may be engineered to have specfic tag
4. When mixed with cell extract target protein should bind to affinity matrix.
5. Beads can then be centrifuged and washed, removing unbound extract components (batch purification)
6. Purified target protein can then be eluted from beads

Question 24

How would you take measurements from gel filtration?

Answer 24

Collect fractions over time-based on size and measure elution volume.

The volume of buffer at which a particular protein exits the column.

Question 25

What factors affect gel separation chromatography?

Answer 25

Size/mass of protein - in effect, the molecular radius, which is generally proportional to mass

The shape of the protein (e.g. globular v fibrous)

Correct gel type as certain resin are specific for their weight ranges

Length of column, longer columns are better for separation

Amount of protein - too much protein can cause broad elution peaks.

Question 26

How does pl this effect protein charge?

Answer 26

When the protein is an environment lower than the iso-electric point it accepts protons becoming more positive (vice versa).

Question 27

What is the principle of ion exchange chromatography?

Answer 27

Sample Loading: The sample mixture is loaded onto a column filled with charged resin beads, either cation exchange (negatively charged) to bind positively charged molecules or anion exchange (positively charged) to bind negatively charged molecules.

Binding: Oppositely charged molecules in the sample are attracted to and bind to the resin, while molecules with the same charge as the resin pass through without binding. The pH of the buffer is carefully controlled to ensure the target molecules have the appropriate charge to bind.

Elution with Salt Gradient: A salt gradient is applied by gradually increasing the concentration of salt in the buffer. The salt ions compete with the bound molecules for binding sites on the resin, displacing them. Molecules with weaker ionic interactions elute first, while those with stronger binding elute at higher salt concentrations.

Collection of Fractions: Eluted molecules are collected in separate fractions, allowing for the isolation of specific proteins or charged molecules based on their binding strength. Each fraction can then be analyzed or further purified as needed.

Question 28

What is the principle of hydrophobic interaction chromatography?

Answer 28

Hydrophobic Surface Binding:

The column resin in HIC has hydrophobic groups (e.g., alkyl or phenyl groups) attached to it. These groups interact with hydrophobic regions on the target molecules.
Proteins and other biomolecules often have hydrophobic regions on their surfaces that can interact with the hydrophobic resin.
High-Salt Buffer for Binding:

When the sample is applied to the column in a high-salt buffer, the salt ions reduce the solubility of hydrophobic regions, exposing them to the resin and encouraging binding to the hydrophobic groups.
This process is known as “salting out,” where high salt concentrations enhance hydrophobic interactions between the protein and the column.
Elution by Reducing Salt Concentration:

To release bound molecules, the salt concentration is gradually decreased. Lowering the salt disrupts hydrophobic interactions, causing proteins to detach from the column in order of increasing hydrophobicity.
Selective Elution:

Proteins with weaker hydrophobic interactions elute first as salt concentration decreases, while more hydrophobic proteins elute later, allowing for effective separation.

Question 29

how is salt conc related to hydrophobicity within HIC ?

Answer 29

As hydrophobicity increases, a larger conc of salt is needed

Question 30

A biotech company is devising a new kit for an enzyme assay. The enzyme catalyses the following reaction.
A + B —-> C+D .
The substrate or the product is not tagged with any specific agent.
The reactant or the products do not absorb or emit light at any specific wavelength. Which of the following assay will be appropriate to measure the rate of a reaction.

Answer 30

Coupled Enzyme Assay

Why: In a coupled enzyme assay, a secondary enzyme is added that reacts with either the substrate or product of the initial reaction to produce a measurable output, such as a color change or a fluorescence signal. Since the substrates (A and B) and products (C and D) in this reaction do not absorb or emit light, a coupled enzyme can provide a measurable change indirectly by converting one of the products into a detectable form.

Question 31

What is a coupled enzyme assay, and how does it measure reaction rates indirectly?

Answer 31

A coupled enzyme assay measures reaction rates by using a second enzyme that reacts with either the original reaction’s substrate or product to produce a detectable signal, such as colour or fluorescence.

This second enzyme’s reaction is dependent on the initial reaction, so tracking its output indirectly reveals the rate of the original reaction.

Question 32

Why are manometric assays only suitable for reactions involving gases?

Answer 32

Manometric assays measure pressure changes in a closed system, which only occur when a gas is produced or consumed.

Therefore, they’re suitable only for reactions where gas changes happen, as reactions without gases won’t show any pressure difference for measurement.

Question 33

In what scenario would a calorimetric assay be appropriate for measuring reaction rates?

Answer 33

Calorimetric assays are useful for reactions that release or absorb a measurable amount of heat.

They’re ideal when the reaction’s heat change is large enough to detect and correlate with the reaction rate, which is often not the case in small-scale enzyme assays that produce minimal heat.

Question 34

What is FRET, and why is it not appropriate for reactions without specific fluorescent or absorbent molecules?

Answer 34

FRET (Fluorescence Resonance Energy Transfer) detects interactions between two fluorescent molecules by measuring energy transfer from one (the donor) to another (the acceptor) when they’re close together.

FRET requires both molecules to have specific absorbance and emission properties, so if a reaction lacks fluorescent molecules, FRET cannot be used to detect or measure it.

Question 35

What considerations are important when selecting an assay method for an enzyme reaction?

Answer 35

Key considerations include the reaction products and substrates’ detectability, whether they emit or absorb light, if heat or gas is produced, the sensitivity required, and if indirect measurement (like a coupled reaction) is needed.

These factors determine the most effective assay for accurately measuring the reaction rate.

Question 36

How can a secondary enzyme in a coupled enzyme assay make a reaction detectable?

Answer 36

A secondary enzyme can convert a product (or substrate) of the original reaction into a molecule that emits a detectable signal, such as a colour change or fluorescence.

This reaction is proportional to the original reaction’s rate, making it possible to measure the original enzyme’s activity indirectly.