lecture 4 Flashcards
What is the ligand in regards to affinity chromatography?
- Attached to an inert matrix , binds only the desired proteins
How can we elute the the protein from affinity chromatography?
Interaction must be disrupted by adding the free ligand or by changing pH of ionic strength
What is the matrix in affinity chromatography?
Its an inert matrix, with an affinity compound attached
What are the 3 different types of biospecific ligand?
- specific
- group specific
- pseudo specific
How can you measure affinity?
Ka and kd are the inverse of each other
What is an example of affinity purification?
- staphylococcal nuclease
How is Staphylococcal nuclease purified?
- binds base analogue
- eluted by change in pH
What are the 3 things you must consider when doing affinity chromatography ?
- protein stability
- Ligand attachment preserves affinity
- elution
What is the ligand modification in affinity chromatography?
spacer to increase accessibility
How can we elute from affinity chromatography?
- use a free ligand
- pH
- Increasing salt concentration
Why would we use affinity tags?
When a protein/ enzyme doesn’t have its own affinity , we can create that affinity using a tag
What is an example of an affinity tag?
HIs tag
What is immobilised metal affinity chromatography?
proteins separated accordingly to their affinity for metal ions that have been immobilised by chelators to an insoluble matrix
What is the normal process for immobilised metal affinity chromatography?
- metal associates and protein with a HIs tag binds , elute with imidazole
What are the advantages of affinity chromatography?
> 95% purity in one step
Tagged recombinant proteins
(His)n
Proteins promote solubility
What is a disadvantage of affinity chromatography?
- Findig an attachable specific ligand
How does gel filtration work?
- forms pores of variable sizes which large proteins are unable to enter .
- Thus elute the larger proteins first
What are the steps needed to perform gel filtration?
- equilibration
- sample application
- elution
How do the proteins elute in gel filtration?
- samples elute in decreasing size, largest comes off first
What is the equation to determine the proportion of pores occupied by a protein?
Kav = (Ve- Vo)/ (Vt- Vo)
What does Vo stand for?
Vo = void volume (outside the beads)
What does Vt stand for?
Vt = total volume
What does Ve stand for?
Ve = excluded volume of a protein
How is the Kav related to the molecular weight of the protein?
Kav proportional to log (MW)
Calibration with known MW
How can you ensure separation of peaks of proteins in gel filtration ?
- range of bead sizes
- increasing the column length
- peak width
What are the advantages of gel filtration?
- simple
- Gentle, conditions as required by protein
Complexes, oligomers Characterisation - Desalting
What are the disadvantages of gel filtration?
- Long columns, time
- Small samples, diluting
Which techniques have a high capacity?
ion exchange, Affinity chromatography and precipitation
Which techniques how a small resolution?
precipitation
Which techniques have a small capacity
gel filtration
Which techniques have a high resolution?
- Ion exchange , Affinity chromatography and Gel filtration
What is the overall best strategy for purifying proteins?
Capture: High capacity
- Precipitation (or IE) end: high ionic strength
Intermediate chromatographic steps
HIC start: high salt, end: low salt
IE start: low salt, end: high salt, concentrated
Polishing
GF start: concentrated, end: dilute
What are the steps to isolate Deacetoxycephalosporin C synthase?
- E.coli lysed by sonication
- Centrifuge ro clarify
- Ion exchange : gradient vs stepwise
- HIC
- GF to polish
What are the steps used to isolate MBP - tagged protein =?
- Maltose - bound column
- Gel filtration
- Analysis y SDS page
What are the steps used to isolate FAB fragment?
- cells lysed with sucrose
- DNAase added
- cation exchange
- HIC pH 5 start 1M (NH4)2SO4
Step to 50% - IE with shallow gradient
What are the 3 ways we can monitor purity?
- specific assays
- total protein concentration
- purification table
What are the 3 types of assays for determinng the purity?
- activity assay
- binding assay
- detection of impurities
What are the advantages of an activity assay?
- tailored to protein
- rapid , reproducible , specific cheap
What are the disadvantages of an activity assay?
often colorimetric , destructive and reduces yield
What are the types of detection of impurities that can be used?
SDS-PAGE
IEF/Western blot/MS
What are the types of monitoring where the total protein is detected?
- absorption at 280 nm
- Bradford assay
Pros vs cons of. the absorption at 280 nm?
pros - Rapid, non destructive
cons - Not quantitative (e unknown), nucleic acids interfere
What are the pros and cons of the Bradford assay?
pros Simple, rapid, fewer interfering substances
cons -Variation in response, requires standard curve (is actually a straight line)
Equation for specific activity
Enzyme activity/unit mass protein
What is the equation for yield?
Total activity after nth step x 100%/ Total enzyme activity in initial sample
What is the equation for the purification factor?
Specific activity after the nth step
/ Specific activity of initial sample
