Structural Biology - Protein Seperation Flashcards

Question

Explain band spreading (diffusion) as a Gaussian shaped profile

Answer 1

highest protein concentration in the middle loss of protein concentration at the edges occurs when column moves too slowly protein concentration vs volume graph Dispertion = volume width of peak at half height resolution = separation of two peaks over the average of dispersed widths

Answer 2

When flow is too fast Solution passes through channels between particles of solid phase. These are uneven so turbulence occurs when flow is too fast

Answer 3

Reduces diffusional broadening Causes turbulent flow and equilibrium may not establish

Answer 4

alpha (the fraction of protein bound to the column) changes as protein diffuses. Lower protein concentration at top and bottom edges of the band = increased likelihood of binding (alpha increases) and slower movement down the column Centre of the band is moving fast (high concentration) and edges are moving slow = sharpening of leading edge of the band and tailing of the rear edge of the band lower resolution in the trailing edge

Answer 5

alpha (fraction of protein bound) increases as protein concentration decreases

Answer 6

Ion exchange chromatography Chromatofocussing Hydrophobic interaction chromatography Affinity chromatography

Answer 7

Hydrophilic to minimise non-specific binding (usually carbohydrate based) highly porous to avoid size exclusion can be derivatised rigid to minimise compression under flow stable to extremes of pH capable of being made into fine particles

Answer 8

A Positively charged group: ex. ammonium. Weak base is protein is highly charged and strong base is protein is slightly charged. A negatively charged group (acid)

Answer 9

Proteins are loaded onto column with low ionic strength. pI is the point where the proteins no charge. Above the pI, protein has -ve charge so binds to a +ve anion exchanger. Below the pI protein has +ve charge so binds to -ve cation exchanger. To release/elute protein, change pH to pI OR increase ionic strength/salt concentration (small ions like NaCl compete for the charge on the protein and column and increase the kD)

Answer 10

Alpha titrates with the ratio of charged (binding) to uncharged (non-binding) groups. Titration of protein groups is more important than the binding sites and this will affect kD

Answer 11

Each favourable charged (ionic) interaction is 1-2kJ mol^-1 ΔG = -RT ln K So each charge of 6kJ mol^-1 alter K by a factor of 10 The same as a change in protein charge of 4 switches between binding and desorption Assume Г = 0.5 mM and Kd = 0.05 mM gives α = 0.91 Changing Kd from 0.05mM to 0.5mM (factor of 10) gives α = 0.5 which allows elution

Answer 12

Fractionation my size and not charge column is porous, so overfilling would close the pores and prevent molecules from moving down the column

Answer 13

pH at the surface may be different from bulk solution, as H+ is excluded and +ve and concentrated at -ve. pH can affect protein stability Use a buffer to minimise pH gradient. But need to make sure that buffer does not bind preferentially hence changing the pH

Answer 14

2 buffers: A (low salt) and B (high salt) flow through valve which introduces the protein and goes into column introduce buffer B high salt over a linear gradient Fraction collector is a series of tubes that collects amounts from column UV detector to measure where the protein is in the fraction collector

Answer 15

A linear gradient of salt is introduced into the column. Rear edge has high salt, front has low salt. High salt = more elution/less binding so increased kD and decreased alpha. so trailing/rear edge catches up with leading edge = improved resolution

Answer 16

Variation of band sharpening but uses pH gradient instead of salt gradient. In a well optimised column resolution is limited by diffusional broadening. try to get the protein to rebind to allow the trailing edge to catch up

Answer 17

Use an anion exchange resin with protein pI 7. pH gradient down the column is very steep (not like salt linear gradient) Column starts at pH 9, so protein binds Elution occurs when pH is close to pI at bottom of column (protein is most concentrated here)

Answer 18

adding acid does not work use exchange and buffer with range of pKa values and constant buffering capacity ex. polyaminopolycarboxylic acids (ampholytes) So proteins elute in order of their pIs

Answer 19

Uses hydrophobic interactions to separate proteins (as proteins with similar pI are difficult to separate) Solid matrix carried hydrophobic groups (phenyl/octyl). Start with high salt and gradient down (opposite to IEC) = salting out effect Highly hydrophobic protein can be eluted with organic solvent/H2O mixture (ethylene glycol) This method is a last resort (IEC should be used first) because it can impact protein stability

Answer 20

Similar to hydrophobic interactions. Solid matrix carries a more hydrophobic ligand like C18 or biphenyl. Under high pressure Gives high resolution Proteins are denatured so not used for soluble proteins (only for peptides/carbohydrates)

Answer 21

Separation based on specific binding between immobilised ligand and protein

Answer 22

non specific binding at high protein concentrations high cost of affinity matrices in industry lower capacity of immobilised ligand (adding too much protein is bad)

Answer 23

Usually used in combination with other methods for 'polishing' steps

Answer 24

substrate analogues/inhibitors for enzymes. cofactors ex. NAD+ lectins - bind glycoproteins Dye ex. blue dye dextran antibodies fusion proteins ex. GST, poly-His, MBP fusion at N or C terminus

Answer 25

CNBr is a pre-activated resin CNBr reacts with OH groups on matrix to form cyanite esters. These groups react with amine groups of ligands to form a covalent bond which immobilises the ligand.

Answer 26

Protein binds to immobilised ligand in the column. Elute by using soluble ligand. Protein becomes more mobile and moves down the column. Works well for lectins and fusion proteins. Can increase concentration of ligand in linear gradient OR in one step. Can also change I (ionic strength) or pH as binding is dependent on these

Answer 27

Polishing method used last due to low resolution and limited capacity. Liquid-liquid phase Column has a range of pore sizes Largest molecules come out first (pass through the side instead of the pores) Water and small molecules come out last (passes through all the pores) No interaction with solid phase (it is inert) No thermodynamic effect (kD) so resolution is low Limited capacity and mixture needs to be concentrated (<10mg/ml) Long thin columns the best

Answer 28

See notes for image V0 = volume outside all the pores, any protein can go through this Vt = total volume Ve = elution volume, where separation occurs

Answer 29

Plots Kav vs log Mr in a straight line Used to buy appropriate column for the Mr

Answer 30

Every step leads to a loss in yield Purification table used to see how efficient each step is Activity is only for enzymes loss of total units but increase in specific activity between each step purification factor used to determine efficiency of each step

Answer 31

Migration of proteins in an electric field Polymeric medium Diffusional broadening still present but reduced as molecules diffuse slower in gel

Answer 32

Field strength (voltage) ionic strength (buffer with higher ionic strength =conducts current more = slower migration net charge temperature molecular size and shape viscocity

Answer 33

Denaturing condition - migration depends on size Native conditions - migration depends on size and charge Isoelectric focussing - migration dependent on pI 2D - migration depends on pI in one dimension and size in the second

Answer 34

Acrylamide cross linked with methylene bis-acrylamide Ratio of acrylamide to methylene bis-acrylamide and concentration determines pore size (which determines rate of separation) 5% acrylamide = no Mw sieving 10-15% = proteins are separated by size

Answer 35

Coomassie blue dye or silver stain

Answer 36

proteins denatured by heating to 100 degrees reduction agent (SDS/meta mercapoethanol) used to reduce disulphide bonds SDS binds hydrophobic parts on protein and unfolds polypeptide SDS is anionic (negatively charged) so now each protein has the same negative charge density proteins migrate according to size

Answer 37

Acrylamide between two plastic/glass plates Anode at bottom, cathode at top Tris-glycine buffer at the top and bottom Top gel (stacking gel) 5% acrylamide pH is 6.7 (All proteins will have the same mobility due to large pores) Second gel (running gel) acrylamide 10-15% acrylamide pH is 8.9 (Molecular sized pores that separate proteins based on size) Smaller proteins run faster than larger proteins

Answer 38

pH 6.7: slightly net negative charge (pI is 6) in stacking gel pH 8.9: large negative charge (above pI) in running gel Rate of migration = V x (δMx) δ is the proportion of charge molecules Mobility is slow when glycine is close to its pI at pH 6.7 Whole population moves as a group (share the small delta negative)

Answer 39

Glycine moves slower than chloride Forms a conductivity gradient (glycine low conductivity+small neg. charge, chloride high conductivity+full neg.charge) Constant current is applied so voltage increases in glycine region Glycine now moves faster Proteins are trapped between two regions Creates a concentrated/narrow protein band reduces human error when pipetting After in running gel: pH increases and glycine is full charged so proteins are sieved by Mr

Answer 40

1. homogenate with proteins 2. IEC more pure sample 3. Affinity chromatography pure protein (should show one band) 4. Marker

Answer 41

Similar to SDS-PAHGE but no denaturing Uses intrinsic charge or protein: most proteins have pI 4-7 and native gels run at pH 8-9.5 so most proteins will have net negative charge Charged dyes can be used The closer the pH is to the pI, the longer the running time

Answer 42

Run denaturing (only size) and non denaturing (separation based on size and charge) at the same time. If the band is shifted up, it is oligomeric. Denaturing will show multiple bands, non-denaturing keeps native interactions so will show a larger band.

Answer 43

Uses intrinsic charge (no denaturing) Migration through a preformed pH gradient across the gel Proteins migrate to their pIs (net charge=0) and focus there (stop migrating) very high resolution <0.1pH units

Answer 44

Ampholytic buffers Are polyomino polycarboxylic acids Have a range of pKa's Have ionisable negative and positive groups Move as a group toward the cathode or anode Separate them out to create a buffer system

Answer 45

Separate hundreds of proteins Migration through a pH gradient first, then rotate field and migrate through SDS gel Get pI vs Molecular weight High resolution

Answer 46

easy and predictable selectivity high separation efficiency small sample sizes fast seperations can be automated and coupled to mass spec quantitation

Answer 47

low concentrations and large volumes difficult for sticky molecules reproducibility problems

Answer 48

Occurs in negatively charged capillary High surface area to volume ratio Electric field is applied along the capillary Many positive charges near negative wall of the capillary Net flow is towards the cathode; even though anions are moving the other way the force of the positive charges is stronger Occurs via electroosmotic flow Molecules migrate according to Mw Data is displayed as an electropherogram

Answer 49

IEF separates proteins based on their pI Electroosmotic flow is suppressed by coating it with teflon Capillary is filled with a buffer to create a pH gradient Electric field is applied Molecules move to pH where their net charge is 0 (pI) and focus there After focusing, zone is mobilised by pressure or chemical means (add salt to compete with H+/OH-)

Structural Biology - Protein Seperation Flashcards

(73 cards)