BB5&6 Protein Isolation & Purification Flashcards
The proteins in solution will denature unless they are stabilized against
- changes in pH (add buffer)
- changes in temperature (cool to 4c)
- protease degradation (add protease inhibitors)
Protein Accessibility
- in solution
- in extra-cellular medium
- intra-cellular = must be broken (lysis)
Protein separation uses differences in
- solubility
- size
- charge
- specific binding affinity
After each step, a test ________ must be performed to see if the desired protein has been separated from the others
assay
Salting out
- uses solubility differences
- different proteins salt out at different salt concentrations
- only partial separation
Solubility changes with addition of
ionic salts
Increasing salt concentration
decreases protein solubility
Uses the size difference between proteins and small molecules to separate them
dialysis
Dialysis uses a
- semipermeable membrane
- dialysis bag submerged in buffer solution without small molecules = diffuse down concentration gradient
- protein molecules retained in the bag
- small molecules pass through the bag into the external solution
Dialysis can
change one buffer for another
• permits changes to be made in the pH of the protein solution
Uses differences in the sizes of proteins
gel-filtration chromatography
Gel-filtration chromatography uses a column of
porous beads of highly hydrated polymer gel
Examples of polymers in beads
- dextran (polycarbohydrate)
- agaraose (polycarbohydrate)
- polyacrylamide
In gel-filtration chromatography, the proteins will flow down through the column and will be collected as
fractions
The flow of … proteins is slowed by the beads, permeates pores of beads
smaller
…proteins flow faster around the beads in the column
larger
Summary of gel-filtration chromatography
- Biggest proteins first through the Beads
- large quantities of proteins can be separated
- separation not well resolved unless there’s a big difference between the sizes
Ultimately uses differences in the masses of proteins
ultracentrifugation
In ultracentrifugation, proteins are separated according to their
sedimentation coefficient
Sedimentation coefficient
- proportional to protein mass
* heavier proteins sediments down faster
Acts against centrifugal force
- buoyancy
* related to protein density
Type of ultracentrifugation
zonal centrifugation
• AKA band centrifugation
• AKA gradient centrifugation
Zonal centrifugation requires … inside the centrifuge tube
density gradient
• suppresses convection currents in the tube
Can be produced by mixing low- and high-density solutions
density gradient
Ultracentrifugation separates the proteins into
bands
• collected as fractions
• heaviest collected first
It is possible to separate proteins by differences in their
surface electric charge
pI – Isoelectric point
value of pH when the net surface charge is zero for that protein
Proteins placed into the gel move within the applied electric field according to their
surface charge
Something is isoelectrically focused when
the surrounding pH equals the pI vale for any given point, protein will cease to move in the field
•high resolution degree of separation
requires a pH gradient gel
isoelectric focusing
• uses a gel of polyampholytes
Polyampholyte
small multi-charged polymers with different values of pI
• applying electric field creates pH gradient
Low pH at
positive (+) anode
high pH at
negative (-) cathode
Proteins have differing numbers of
acidic and basic residues
• often have an overall positive or negative charge
Uses beads with a surface charge chemically attached to them
ion-exchange chromatography
• beads typically cellulose or aragose
Negative beads
Carboxymethyl – CM cellulose
Positive beads
Diethylaminoethyl – DEAE cellulose
Proteins with positive charge attach to
negatively charged beads
• other proteins pass down the column unhindered
Positively charged proteins can hen be … from the column
eluted
eluting = releasing
Eluting bound proteins
- add low concentration of salt (sodium highly positive)
- sodium ions bind to the beads instead of the proteins
- weakly positive proteins elute off first
- increase salt concentration = more positively charged proteins elute from column
- all positively charged proteins can be collected as fractions as they come off the column
Separates proteins according to size by applying an electric charge through a polymer gel
gel electrophoresis
PAGE
Polyacrylamide Gel Electrophoresis
• keeps from “falling down”
• forms spaghetti-like strands
Most common forms of gel electrophoresis uses
Sodium Dodecyl Sulphate – SDS
SDS-PAGE
SDS is an
anionic detergent
• disrupts all non-covalent interactions
• binds to amino acid residues in 1SDS : 2amino acid
• all proteins become negatively charged
The negative charge on each protein becomes directly proportional to its
mass
Beta-mercaptoethanol added to disrupt
disulfide bonds
• proteins become fully denatured
Protein mixture flows down the gel…
from the cathode toward the anode
• large proteins impeded in the gel by the strands
• Smallest proteins fastest through the Strands
In gel electrophoresis, the protein separation provides a direct measurement of their
masses
• proteins with known molecular masses run as a scale marker
Makes use of the fact that many proteins tightly bind small specific molecules as part of their function
affinity chromatography
…binds glucose very tightly
Concanavalin A
Affinity chromatography overview
- covalently attach glucose to beads in a column
- pass crude protein mixture w/ Concanavalin A down the column
- Concanavalin A will bind to the glucose on the beads
- all remaining proteins pass through unhindered
Concanavalin A can be removed by passing … down the column
concentrated solution of glucose
• binds better to “free” glucose than to the “bound” glucose on the beads
•concanavalin A will elute from the column bound to the free glucose
• the free glucose would be removed by dialysis
Specific activity
Enzyme activity & protein concent
• the ratio of enzyme activity to the amount of protein in the mixture
Forming homogenate with centrifuge
- dense pellet at bottom
* light supernatant at top
Deferential centrifugation
Yields several fractions of decreasing density, each with hundreds of different proteins
• to homogenate
Eluting proteins in ion-exchange chromatography
- increase concentration of sodium chloride ( or other eluding buffer)
- compete with positively charged groups on the protein for binding to the column
- low density of net positive charge emerge first
- followed by those with a higher charge density
Cation exchange
(Ion exchange chromatography)
• positively charged groups bind to anionic beads
Cationic proteins can be separated by chromatography on
Negatively charged carboxymethylcellulose
(CM-cellulose) columns
Anionic proteins an be separated by anionic exchange on
Positively charged diethylaminoethylcellulose (DEAE-cellulose) columns
