Protein purification Flashcards
What does Native PAGE stand for?
Native Polyacrylamide Gel Electrophoresis.
Name some sources of proteins
-Native proteins (extracted from the host organism)
-Recombinant proteins (heterologous protein expression systems).–> gene cloned in a vector and inserted into the organism that produces the protein.
What characteristics separate proteins?
-Size (size exclusion chromatography)
-Charge (ion exchange chromatography)
-Specific Group (affinity chromatography)
-Hydrophobicity (hydrophobic interaction chromatography).
What factors influence the relative migration rate of proteins in Native PAGE?
The relative migration rate is based on both the protein’s mass and charge at the pH used.
Name 2 features in common for each chromatography
-Stationary phase (immobilisation of sample)
-Mobile phase
How do molecules move in an electrical field during electrophoresis?
Anions (negatively charged) move towards the anode (+), while cations (positively charged) move towards the cathode (-).
What is the role of Sodium Dodecyl Sulfate (SDS) in SDS-PAGE?
SDS denatures proteins and imparts a uniform negative charge, allowing separation based on size.
What must be done to disulfide bonds before SDS-PAGE?
Disulfide bonds must be reduced using a reductant like dithiothreitol (DTT) or β-mercaptoethanol (β-ME).
What does the term ‘Isoelectric Focusing (IEF)’ refer to?
A technique used to separate proteins based on their overall charge.
What is the purpose of 2D gel electrophoresis?
To analyze complex mixtures of proteins by separating them in two dimensions: first by charge (IEF) and then by size (SDS-PAGE).
What is proteomics?
The study of the complete set of proteins expressed by a genome, including their functions and interactions.
Why is protein purification necessary?
-To characterize proteins, investigate functions
-study post-translational modifications
-use in diagnostics or commercial applications.
What are the main characteristics used for protein separation?
Size, charge, specific groups, and hydrophobicity.
What is the principle behind Size Exclusion Chromatography? What elutes first?
It separates proteins based on size, with larger proteins eluting first.
What is the Isoelectric Point (pI) of a protein?
The pH at which the protein has a net charge of zero.
What is PI similar to?
pka
In Ion Exchange Chromatography, how are proteins separated?
By their charge, using positively or negatively charged matrices.
What is the role of hydrophobic interaction chromatography?
To separate proteins based on their hydrophobicity, where proteins interact with hydrophobic beads in the presence of high salt.
How can proteins be engineered for purification?
By adding tags or sequences that facilitate detection, folding, or secretion.
What is Mass Spectrometry used for in protein analysis?
To determine the accurate mass of proteins and can provide amino acid sequences and structural information.
What is the significance of using a His-tag in protein purification?
It allows for efficient purification via affinity chromatography, specifically binding to metal ions.
What does Size Exclusion Chromatography separate based on?What is eluted first and why?
-size
-larger proteins eluted first because smaller proteins are more likely to get lost
Name some factors that have an influence on the size of peaks for absorbance
-Number of Tryptophan groups in the protein
-Volume of overall protein
What is Hydrophobic Interaction Chromatography used for?
to separate proteins based on their hydrophobicity
