Protein Purification Flashcards
What is protein purification?
Multi-step fractionation process to isolate a single protein from a crude mixture
What is the most challenging job as a biochemist?
Protein purification
What was the first enzymes purified and crystalized?
Urease and Pepsin
What are the steps for protein purification?
- Cell lysis
- Differential centrifugation
- Salt fractionation
- Dialysis
- Ion-exchange
- Gel filtration
- Affinity
- Concentration
- Purified protein which will go through structural and functional analysis
What is cell lysis?
Disruption of cells
Physical methods - mechanical shearing
Chemical methods - solubilizing plasma membranes
Enzymatic methods - cell wall digestion
What is cell fractionation?
Using centrifugation (applying centrifugal field), will isolate high density proteins into a pellet and low density protein as the supernatant
Size and density of particle, duration of centrifugation
Bacteria has less organelles so fractionation is simple but eukaryotic have many organelles.
What is differential centrifugation (subcellular fractionation)?
RPM between small and large will stay constant which is bad but RCF will change.
RCF important factors:
- angular velocity (speed)
- average radius
What is zonal centrifugation?
Fractionate into bands
- the large particles rapidly sediment
- the more spherical particles rapidly sediment
- influenced by the density of particles and the medium
vacuoles never reach bottom because equal density
What is salt fractionation?
- Protein dissolved in water
- proteins are soluble because it is polar and makes hydrogen bonds - Salt (precipitate and concentrate protein)
- Protein in ammonium sulfate
- water divided from protein to ions
- need to aggregate with each other to bind
Less soluble proteins require how much % of salt saturation?
Less soluble proteins require less % of salt saturation.
What is the ammonium sulfate precipitation equation?
Weight of salt (g) = Gsat (S2-S1) / 1-(PS2)
What is dialysis?
A separation technique of removal of small molecules from a protein mixture using semipermeable membrane
What are the uses of dialysis?
Desalting
- removal of salt and many other small molecules from a protein mixture
Removal of small peptides and proteins
- anything under molecular cut-off (MWCO) such as 10 kDA will leave the bag
- anything above will stay inside the bag
Change to fresh buffer to remove more salt due to osmosis
Is changing the buffer multiple times better for dialysis?
Yes
What are the positively charged amino acids?
Lysine (Lys, K)
Arginine (Arg, R)
Histidine (His, H)
What are the negatively charged amino acids?
Aspartic acid (Asp, D)
Glutamic acid (Glu, E)
When a protein is coated with positive charges, which ion exchange to use?
Use a carboxymethyl (CM) sepharose which is a cation exchanger with negatively charged beads
When a protein is coated with negative charges, which ion exchange to use?
Use a diethyl amino ethyl (DEAE) sepharose which is a anion-exchanger with positively charged beads
What are the three properties that affect ion-exchange chromatography?
Charge state of peptide of protein
pH of buffer
pI value, need to be 2.5 pH higher or lower than pI
What is cation exchange chromatography?
Negative charged beads in the column
Negative charged proteins are collected in the flow through
Positive charged proteins will bind to the beads
Elution buffer is used to elute the bounded proteins
How does separation of proteins depend upon net charge?
With an anion-exchange chromatography (DEAE, + beads), the most positive charge will elute first as it won’t bind and the most negative will tightly bind and elute last
What is required to elute proteins?
Salt
Protein binds at low ionic strength and elutes differentially by increasing salt concentration
What is gel filtration?
Separation based on size (molecular weight)
Largest molcules are excluded from the beads and elute first
Smallest molecules are included in the beads and elute last
What is elution volume (Ve)?
the volume of a buffer required to elute a compound from the time of its injection to its exit from a column
Measures how well a compound interacts with a stationary phase
What is the retention factor (K’)?
K’ = Elution volume of a compound (Ve) / Elution volume for a solute which does not interact with stationary phase (void Vo)
What can the retention factor (K’) determine?
Determine the molecular weights of unknown proteins
What is resolution?
Ability of a column to separate two components (proteins) wide apart
Peaks elute at different volume and do not overlap
Resolution equal or higher than 2 is considered significant
Higher resolution means better separation
What does resolution depend on?
Particle size
Flow rate
Column length and diameter
Sample volume
