Flashcards in Protein Purification and Analysis Deck (92):
What are the 4 levels of protein structure?
Primary, secondary, tertiary, quaternary
Define primary structure of a protein
Linear amino acid sequence of the polypeptide
Define secondary structure of a protein
Local structure of linear segments of the polypeptide backbone atoms
Define tertiary structure of a protein
Three-dimensional arrangement of all atoms
Define quaternary structure of a protein
Arrangement of separate polypeptide chains into the functional protein
Why do we purify proteins?
Which proteins are involved in the process?
What does each protein do?
How does it do it?
Can we interfere with its function (therapeutics)?
Because functional studies are difficult in cells
To study a protein in vitro, what do we need large amounts of?
Natural sources (tissues, bacteria, plants)
Recombinant protein expression (through cloning)
Which organisms can we clone proteins into to obtain overexpressed proteins?
Bacteria (E. coli)
Insect cells (Baculovirus)
In basic terms, how do we purify a protein?
Kill the organism, lyse the cells, purify the protein
What key molecule has been investigated using pure protein?
The making of ATP synthase
Does colocalisation of proteins mean interaction?
What methods can we use to look at the structural biology of proteins?
Nuclear magnetic resonance (NMR)
What can we do with pure proteins?
Look at the structural biology
Carry out binding/enzymatic/functional assays
What is mass spectrometry?
A very accurate method of molecular weight determination and identification of proteins
What does a Western blot require?
An antibody specific for the protein of interest or for the affinity tag
Briefly outline the steps used to carry out a Western blot analysis
1. Run an SDS-PAGE gel
2. Transfer to nitrocellulose
3. Incubate with specific antibodies
5. Western blot
What is SDS?
A detergent which binds proteins non-specifically
What does SDS stand for?
Sodium dodecyl sulphate
What does SDS do to proteins?
It linearises them and coats them in a negative charge
What does PAGE stand for?
Polyacrylamide gel electrophoresis
Why is it necessary for the SDS-PAGE step when purifying proteins?
To separate proteins by their mass
What are absorbance measures for detecting proteins useful for?
Quickly screening which purification fraction contains proteins
List some direct ways of detecting a protein of interest in a mixture
List some indirect ways of detecting a protein of interest in a mixture
Using specific properties of the protein
What is GST?
A protein of 25kDa that binds Glutathione with high affinity
How do you purify proteins using GST affinity chromatography?
Elute protein with a gradient of free reduced Glutathione which competes with immobilised Glutathione for binding GST
What is FLAG?
A small peptide that binds specifically to Flag antibody with high affinity
How do you purify proteins using FLAG affinity chromatography?
Elute protein containing a Flag tag with free FLAG peptide
Many different types of affinity chromatography tags have been developed and are commercially available, give examples of the most common ones and what they bind to
Hexa-histidine (His6) binds to nickel-NTA
GST binds to glutathione
FLAG binds to an anti-Flag antibody
How do you purify proteins using hexa-histidine affinity chromatography?
Elute protein with a gradient of Imidazole which competes with histidine for Ni2+ binding
What is the matrix made up of in hexa-histidine affinity chromatography?
What do histidine amino acids have a strong affinity for?
Nowadays, how are the majority of proteins expressed?
What is the most common method of protein purification considering that the majority of proteins are expressed recombinantly?
What does the level of purity achieved from affinity chromatography depend on?
The tag and protein of interest
It is often necessary to do a 2nd purification step when purifying proteins using affinity chromatography, give examples of these 2nd step purification methods
Ion exchange chromatography
Size exclusion chromatography
What method is only possible when using recombinant protein expression?
What do many vectors contain that is useful for affinity chromatography?
An affinity tag
How is the protein released in hydrophobic interaction chromatography (HIC)?
By lowering the ionic strength
What does the matrix in HIC consist of?
Beads of hydrophobic polymers
How are proteins separated in HIC?
By their hydrophobicity (solubility)
In HIC, what do the hydrophobic polymer beads bind?
Hydrophobic amino acids of proteins at high ionic strength
What is the purification of proteins based on?
The physical properties of them, e.g. molecular weight, isoelectric point, solubility
One purification methods is sufficient to obtain a pure sample of protein, true or false?
False, generally need to combine many purification methods such as ion exchange and size exclusion chromatography
What does a protein's hydrophobicity profile depend on?
The surrounding salt concentration
Many amino acids are hydrophobic, but where do these amino acids tend to be located within the protein structure?
On the inside
What is the range of molecular weight of proteins?
From 2kDa up to 3.8 million Da
How is molecular weight expressed?
In Dalton (Da)
What does a protein's molecular weight depend on?
The number of atoms (amino acids)
What is 1Da equal to?
~1.66 E-27 kg
At a protein's isoelectric point, what is the number of positive charges equal to?
The number of negative charges
At pH values below the isoelectric point, is the protein positive or negatively charged?
Define isoelectric point (pI)?
The pH at which the protein's net charge is 0
What is the charge on an amino acid at neutral pH?
Some amino acids are negatively charged, some are positively charged
What do the physical properties of proteins depend on?
The primary sequence
What does the choice of a protein expression system depend on?
The properties of the target protein
The type of analysis post-purification
What do genes encode?
What are proteins crucial for?
How many genes do humans have?
How many proteins do humans have?
Linear chains of amino acids which fold into a specific structure depending on the sequence
Is the backbone of an amino acid common or different in every amino acid?
Common to all amino acids
Is the side chain of an amino acid common or different in every amino acid?
Different in every amino acid
What is protein structure and function defined by?
Amino acid properties
What types of properties can amino acids have?
Large molecules in size exclusion chromatography cannot enter the pores in the matrix so what happens to them?
They move rapidly through the column
Small molecules in size exclusion chromatography can enter the pores in the matrix, how does this reflect the time these molecules to take to move through the column?
They take longer
What is size exclusion chromatography otherwise known as?
Molecular sieve or gel filtration chromatography
What is the matrix of size exclusion chromatography made up of?
Porous beads of carbohydrate polymer, e.g. dextran, sepharose or agarose
What is the matrix of ion exchange chromatography made up of?
Beads of polymers with a charged functional group
How does size exclusion chromatography separate proteins?
By their size (molecular weight)
How does ion exchange chromatography separate proteins?
By isoelectric point
What charge are anion exchange resins in ion exchange chromatography and what do they bind?
Positive and bind negatively charged proteins
What charge are cation exchange resins in ion exchange chromatography and what do they bind?
Negative and bind positively charged proteins
Simply, how does ion exchange chromatography work?
The column binds proteins with the opposite charge
How are proteins purified in ion exchange chromatography?
Salt competes for ion binding sites and allow proteins to be released
Typically use a gradient, e.g. 0 to 1M NaCl
In ion exchange chromatography, what happens the greater the charge on a protein?
The tighter it binds the resin, the more salt is needed to dislodge it and the later it elutes
The charge on a protein is affected by pH, and so what protein purification method is very sensitive to pH?
ion exchange chromatography
What does the choice of protein purification method depend on?
The protein of interest: size, charges, hydrophobicity
Before starting protein purification for analysis, what is it important to analyse first and why?
The amino acid sequence of the protein to help design an optimal purification stategy
What is the maximum absorbance value of the tryptophan chemical group?
What is the maximum absorbance value of the phenylalanine chemical group?
What is the maximum absorbance value of the tyrosine chemical group?
What is the maximum absorbance value of the peptide bond chemical group?
How is absorbance used to detect proteins?
As a general method to use the UV absorbing properties of proteins
At what wavelength do most proteins absorb at and what is the disadvantage to this?
Doesn't allow the discrimination between your protein of interest and the contaminants
Some proteins have activities or properties that can be analysed in the test tube (in vitro), true or false?
Do proteins have cellular functions?
Anything that the protein of interest does that distinguishes it from others can be used for what?
An assay, i.e. a method to detect its presence
Give examples of indirect methods for detecting proteins
Detecting metalloproteins by using the properties of the metals
When using pure proteins, you can test the direct interaction between 2 proteins using binding assays, name 2 of examples of these types of assay