Chromatography and Protein Analysis Flashcards
List five features by which proteins differ.
Size
Charge
Hydrophobicity
Stability
Isoelectric point (pI)
What must generally be performed on a protein, prior to analysis?
Purification
Protein biopharmaceuticals, such as insulin, must be ______ for use.
Pure
True or false: if pH is greater than pI, the protein will be negatively charged.
True
If pH is _______ than pI, protein will be positively charged.
Greater
Column chromatography is a _____________ method.
Separation
In column chromatography, the stationary phase is usually _________.
Solid
The ___________ phase is usually liquid or gas, flowing over the stationary phase.
Mobile
____________ takes place based on the material’s interaction with the stationary phase, relative to its interaction with the mobile phase.
Separation
Material used determines the ability of the column to withstand ___________.
Pressure
Gel filtration is an example of ____________ chromatography.
Partition
______________ chromatography includes ion exchange and hydrophobic interactions.
Adsorption
What is affinity, in the context of protein separation?
The ability of many biomolecules (particularly proteins) to recognise specific chemical shapes and structures
A small molecule or ‘___________ ________’ is immobilised in a solid stationary phase.
Affinity ligand
Outline the principle of affinity chromatography.
A small molecule or ‘affinity ligand’ is immobilised in a solid stationary phase. The mobile phase containing the protein of interest is passed over the stationary phase. Interaction between the protein and the ligand causes protein to bind to the column. Impurities are washed out of the column. Elution may be achieved by lowering the pH, or adding a competitor molecule. Less commonly, ionic strength elution, or chaotropic agents (such as urea or guanine hydrochloride), can be used to caused disassociation of the ligand
How may elution be achieved?
By lowering the pH, or adding a competitor molecule
What is a ligand?
A molecule that binds reversibly to a specific molecule, or group of molecules
Why must a ligand have chemically-modifiable groups?
To allow it to attach to the matrix, without destroying its binding activity
What is a spacer arm used for, and how does it work?
To improve binding between the ligand and target molecule, by overcoming any effects of steric hindrance
What is ligand coupling?
The covalent attachment of a ligand to a suitable pre-activated matric, to create an affinity medium
Metals, such as copper, cobalt, and zinc (all divalent) can be immobilised in the solid stationary phase, bound to agarose, in ____________ ________ ___________ chromatography.
Immobilised metal affinity
Proteins bind to __________ via histidine, cysteine, and tryptophan.
Metals
True or false: best binding is achieved in the presence of acidic conditions, with 0.5-1.0 M NaCl.
False
In immobilised metal affinity chromatography, how can elution be achieved?
Via changing pH, addition of EDTA, or adding competitor molecules
____________ binds to nickel or other divalent cations, through its nitrogen.
Imidazole
Following chromatography, the proteins isolated can be visualised using a technique called sodium ___________ sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
Dodecyl
Proteins are studied using vertical gels, made of ________________, which acts like a molecular sieve.
Polyacrylamide
An ___________ ________ is applied, and proteins, negatively-charged after SDS treatment, move.
Electrical field
_____ is a denaturant that unfolds proteins, linearising them, to prevent shape affecting movement, by disrupting hydrogen bonds.
SDS
SDS binds to the unfolded proteins, and gives them an overall _____________ charge, masking the protein’s own charge.
Negative
True or false: smaller proteins are run on higher-concentration gel.
True
____________ proteins are run on lower-concentration gel.
Larger
Describe the procedure of SDS-PAGE.
Loading dye is added to sample
The gel is run, and various buffers can be used
Two different buffers are within the buffer (tris-glycine/HCl at pH 6.8 and pH 8.8). Leading buffer ion (chloride) is in the stacking gel; trailing buffer ion (glycine) is in the upper gel tank
The cast is opened
Washing, staining, then washing again is performed
Visualisation is performed
After electrophoresis, _________ bands are resolved.
Discrete
Coomassie Brilliant Blue is sensitive to ____ µg of protein.
0.1
________ staining is sensitive to 1 ng of protein.
Silver
