Lecture 4 Flashcards
What happens at pH 2.5?
a-amino groups exist as NH3+, while a-carboxylate groups exist as 50% COO- (deprot) and 50% COOH (protonated) giving the amino acid an overall positive charge.
What does the exact value of overall charge depend on?
Specific pKa values of the various groups in each amino acid.
What does the size of net charge determine?
How tightly each amino acid binds.
What does high Na+ present in elution buffer first displace?
Weakly bound amino acids
What happens as [Na+] is increased?
More tightly bound amino acids are progressively displaced
What will an increase in pH lead to?
To eliminate the positive charge on the amino acid, so it no longer binds to then resin.
What are proteins derived from?
Microbial cultures, plants or animal tissues such as liver.
What is crude extract?
The cells are broken open to release the proteins into a solution.
What is separation by ion exchange based on?
Charge differences among proteins.
What is charge differences among proteins based on?
The relative number of Asp and Glu (negative) versus His and Lys and Arg (positive) in each protein and on pH
What is the percentage of proteins negatively charged at pH 7?
~65%
In affinity chromatography, what is the chemical group that is covalently attached to the beads in the column?
Ligand
What happens to the proteins that have an affinity towards the ligand?
Bind tightly to it
What happens to the other proteins that don’t have an affinity towards the ligand?
Move faster down the column and are eluted from the column.
How are the bound proteins eluted?
By the addition of high concentration of salt (weakens the binding between the ligand and the proteins) or ligand (competes with attached ligands)
How can an ATP binding protein be isolated?
By attaching a molecule that resembles ATP to the beads.
What can Genetic Engineering do?
Fuse a “Tag” to the target protein allowing purification by affinity chromatography.
What is a Tag?
A peptide or protein that binds a ligand with high affinity and specifically that is fused to the gene encoding target protein.
In metal affinity chromatography, what do clusters of His in a protein bind tightly to?
Ni2+ and Co2+
What is a His tag?
6 - 8 extra His residues are fused to the target protein at N- or C- terminus.
What is the column made up of?
Chelating resin containing Ni2+
What do His-tagged proteins bind to?
Tightly to the Ni2+ resin.
How is the bound protein eluted?
Adding imidazole (similar to His side chain) to the buffer which outcompetes His-tag and protein no longer binds to the column.
What does high purification in one step cause?
Affect the properties of the protein.
The tag can be removed once the protein is isolated.
What does Gel Filtration or Molecular Exclusion chromatography do?
Separates proteins on the basis of size using beads of POLYMERIC GEL - a loose network of polymer with many water filled pores.
- Larger proteins are excluded from the pores
- Protein molecule can enter the pores if they fit.
What molecules elute first using Gel Filtration chromatography?
Larger proteins elute first and smaller proteins elute later.
What can Gel Filtration be used for?
To measure the molar mass of proteins.
What can proteins be separated and characterized by?
Electrophoresis (NOTE: will never use this method to isolate a protein)
What can electrophoresis be used to estimate?
- Number of proteins in a mixture
- Degree of purity of a mixture
- Isoelectric Point
- Approximate Molecular Weight
In electrophoresis, what does the rate of movement depend on?
Size, shape and change.
What polymer is electrophoresis of proteins carried out in gels made up of?
Polyacrylamide: has right porosity for proteins (10kDa - 1000kDa)
What are the components of a typical gel?
5 - 15% polymer and 90 - 95% water with conductive buffer.
How are separated proteins visualized?
Adding a dye such as Coomassie blue which binds to proteins
What does SDS do?
Binds to proteins and partially unfolds them
What is SDS?
Sodium dodecyl sulphate, which is a detergent.
What do the sulphate moieties of the SDS molecules confer
A large net negative charge on the protein swamping out the native charge
All proteins have a:
Uniform charge per size
What is separation based strictly on with SDS?
Size
In SDS what do smaller proteins do?
Migrate faster than larger ones.
What can you do by comparing the positions to which proteins of known molecular weights
