LAB(4) - PROTEIN ISOLATION TECHNIQUES Flashcards
Protein purification
a series of processes intended to isolate one or few protein form a complex mixture, usually cells, tissues or whole organisms
an essential 1st step in understanding their function
proteins must be released from the cell to be purified
based on the basic properties of protein like solubility (salt, pH, temperature), size, charge, and binding properties (ligands)
protein purification
What are the general steps in protein purification?
- selection of a protein source
- tissues homogenization and solubilization
- stabilization of proteins
- isolation techniques utilize different properties of protein
Homogenization
the process requires the disruption of the outer cell membrane,
intracellular membranes and the surrounding
extracellular structures.
(huh?) For proteins from muscle that would mean grinding it up, for an intercellular protein that would mean breaking the cells open, etc. This is
always done in the presence of a buffer and inhibitors.
What does the use of detergent solution do?
increase the
solubility of proteins (for protein membrane
extraction). Help stabilize and solubilize
proteins as they are released from the cell.
What does the use of other reagents (chaotropic agents) such as urea and guanidine HCl (they breakdown the structure of the protein and dissolve well in water) do?
Help break apart the lipid cell membrane
Salting in
refers to the increase of proteins
solubility in a solution with low salts concentration.
Low salts concentration, solubility of protein increases.
Salt molecules stabilize protein molecules by?
Decreasing the electrostatic energy between the
protein molecules which increase the solubility of
proteins. (salting in)
Salting out
the precipitation of the
proteins at high salts concentration. It is a
purification method that relies on the basis of protein solubility (reducing the solubility)
High salt concentrations
- increase ionic strength of a protein solution
- decrease protein solubility thus precipitation
a purification method at initial molecule purification that lacks the ability for precise
isolation of a specific protein.
Salting out
Ammonium sulfate
commonly used salt
Why ammonium sulfate?
- large solubility in water
- relative freedom from temperature effects
- no harmful effects on most of the proteins
The amount of salt needed to isolate a specific protein is determined from the salt’s fractionation table
study the Hofmesiter series !!!
the proteins are separated after salt addition by?
Centrifugation
Centrifuging the liquid containing the cells in a high-density medium may precipitate the desired cells depending on the density of each constituent cell.
additionally
applicable for eliminating undesired cellular impurities or obtaining certain cell organelles.
Density-gradient ultracentrifugation
Dialysis
Removal of salt
molecules from the
isolated protein solution through a semi
permeable dialysis bag
The salt molecules move from the more
concentrated solution
(from inside the dialysis bag) to the less
concentrated solution.
Buffers and salts exchange until an equil. is established b/w the inside and outside if the membrane; this process could not distinguish b/w proteins effectively.
Chromatography
a separation technique used to separate complex mixtures of amino acids.
Proteins are separated using column chromatography
(..in particular high-performance liquid
chromatography (HPLC)) because?
proteins tend to
denature when separated using TLC.
What are the advantages of HPLC over TLC?
- Resolution and sensitivity
- Quantitative analysis
- Selectivity and versatility
- Automation and reproducibility
- Speed and throughput
Chromatography
based on the principle where molecules in mixture applied onto the surface or into
the solid, and fluid stationary phase (stable phase) is
separating from each other while moving with the aid
of a mobile phase
one of the most powerful fractionation methods.
Column chromatography
Column chromatography can separate components o mixtures based upon?
- adsorption (liquid-solid)
- partition (liquid-solid)
- affinity
- differences among their molecular weights
Gel filtration chrom. (GFC)
a form of partition
chromatography used to separate molecules of different molecular size.
GFC:
- small molecules = freely enter internal solvent space of the gel bead
- larger molecules = too large to penetrate the gel pores and travel b/w beads and elute first
Ion exchange (by charge)
Proteins may have net positive or negative charge at different
pH range. If a protein has a net positive charge, negatively
charged molecules bind to positively charged solid supports
and positively charged molecules bind to negatively charged
Anion exchange resins (positive charge)
separate negatively charged compounds
Cation exchange resins (negative charge)
separate positively charged compounds
Affinity chromatography (by specific binding affinity)
a separation method based on a specific binding
interaction between an immobilized ligand and its binding partner.
Example enzyme/substrate,
receptor/ligand, enzyme/inhibitor,
antibody/antigen interactions.
Affinity chromatography (by specific binding affinity)
High selectivity, resolution, and capacity in protein
purification schemes can be easily achieved with affinity chromatography.
How does affinity chrom isolate targeted proteins?
- attaching the chemical grp. which could affinity with target protein to the column
- Loading the sample into the column and let the target protein affinity with chemical grp.
- eluting the target protein by high concentration
Affinity chrom.:
- the ligand is immobilized onto a solid support matrix
- the crude extract is passed through the column
- the target molecule for which the ligand possess affinity is retained
- all other material is eluted
- the bound target protein is eluted by alteration of the mobile-phase conditions
High performance liquid chromatography
a process of separating components in a liquid
mixture. A liquid sample is injected into a stream of solvent (mobile phase) flowing through a column packed with a separation medium
(stationary phase).
High performance liquid chromatography
- The flow rate of the solvent is set through
computer input and controlled by pumps. - The peak represent different kinds of compounds and result is high resolution and rapid separation
study on the HPLC flow diagram, overview,
HPLC:
Once in the column, the sample mixture separates as a result of different components adhering to or diffusing into the gel in form of different zones called “bands”
Mass spectrometry
used to identify
unknown compounds via molecular weight
determination, to quantify known compounds,
and to determine structure and chemical
properties of molecules.
Edman degradation
classic method to
sequentially removes one amino acid at a time from the N-terminus of a protein, which is then
identified.
Electrophoresis
a separation technique used to separate proteins based on charge and size by applying an electric field
Protein electrophoresis
typically carried out in a gel matrix
Polyacrylamide gel electrophoresis (PAGE)
carried out in a gel which serves as a molecular sieve
study the two forms of electrophoresis
SDS-PAGE
- protein mixture is heated in the presence of 2-mercaptoethanol and SDS
- unfolded polypeptide chains will then migrate towards the anode
- smaller polypeptides migrate further through the gel than larger one
Proteins are purified from a mixture using combination of several techniques based on protein properties.
protein property = technique
solubility = differential precipitation
molecular size = gel filtration chromatography
molecular charge = ion exchange chromatography
hydrophobicity = reversed phase HPLC
biological activity = affinity chromatography
Trivia:
- The first breakthrough in speeding up protein synthesis
was the development of a device in 1960 by R. Bruce
Merrifield that could automatically synthesize
polypeptide chains. - Pancreatic ribonuclease (124 amino acid units) and
human growth hormone (188 amino acid units) have
been prepared with this technique - Biological systems ( i.e. via genetic engineering) are
currently being used and developed to speed up the
process of protein synthesis.
