Techniques Flashcards
1
Q
NMR
A
Nuclear Magnetic Resonance
- Used for solving protein structure
- Needs a (relatively) small protein
- Provides info on distances between atoms
- Good for studying dynamics in solution
- Not the most predominant method
2
Q
X-ray Crystallography
A
- Used to solve protein structure
- Need a crystal (which is difficult to form esp. when its a big protein)
- Get a static snapshot
- High resolution
- Most prominent method
3
Q
Cryo Electron-Microscopy
A
- Used to solve protein structure
- Need a (relatively) big protein (good solution for when its tough to form crystals)
- Directly captures images of proteins in a thin layer of buffer using an electron microscope
- Tech improvements means it is rapidly become the norm
4
Q
Alpha Fold
A
- Program for predicting protein structures
- Gives you confidence scores for the fold predictions (color coded, red = low confidence, blue = high confidence)
5
Q
pLDDT
A
Predicted local distance difference test
- helps you know if model is good
- gives overall confidence in sequence model
6
Q
PAE
A
Predicted aligned error
- Helps you know if model is good
- confidence in alignment of model
7
Q
Denaturants
A
How we study protein folding; denaturants are a way of changing folding
- Chaotropic salts
- Consideration of disulfide bonds
- heat
- Organic solvents
- Detergents
8
Q
Chaotropic Salts
A
Denaturant
- Chemical denaturant that makes bonding less favorable
- Interferes with hydrogen bonding networks
- Increases the solubility of non-polar molecules in H2O
- e.g. urea and guanidine
- need to use high concentrations (ex. 8M of urea)
9
Q
Management of disulfide bonds
A
Addition of reducing agent β Mercaptoethanol (βMe) breaks covalent disulfide bonds
- DDT has two sulfur groups (βMe only has one) and therefore has more reductive power
10
Q
Protein purification
Methods of cell disruption
A
- chemical methods
- enzymes
- structural damage
11
Q
Protein purification
Methods of Debris removal
A
helps remove insoluable cellular components
- centrifugation
- filtration
12
Q
Protein purification
Methods of Initial purification
A
- heat denaturation
- salts
- solvents
13
Q
Protein Purification
Methods of High Resolution Purification
A
- ion exchange
- size exchange chromatography
- affinity chromatography
- electrophoresis
14
Q
What method of purification should you used based on the characteristic
Solubility
A
Salting out
15
Q
What method of purification should you used based on the characteristic
Ionic Charge
A
- Ion echange chromatography
- Electrophoresis
- Isoelectric focus
16
Q
What method of purification should you used based on the characteristic
Size
A
- Size exclusion chromatography
- SDS-PAGE
17
Q
What method of purification should you used based on the characteristic
Binding specificity
A
Affinity Chromatography
18
Q
Steps of
Cloning the gene of interest into a plasmid
A
- Restriction enzyme recognizes restriction site and cuts DNA into fragments with sticky ends
- Addition of DNA fragment from other source using sticky ends to connect plasmid to insert through base pairing
- DNA ligase anneals the strands
== Recombinant DNA molecule
19
Q
Transformation and Selection
A
- DNA from ligation + bacteria in tube
- heat shock tube to create pores in plasma membrane that allow plasmid through
- Grow in a medium with antibiotic to select for bacteria with the plasmid incorporated
20
Q
Things to consifer when choosing a vector
A
- Selectable marker (ex. antibiotic resistance)
- Copy number (# of plasmid copies expressed)
- Promoter
- Method of induction (how you turn on expression, wait until culture is grown)
- Fusion protein (ex. fuse to his tag)
- Tag removal (tag can effect function, how will you remove, ex. protease)
- Are post-translational modification important? (does E. coli have necessary machinery to add mod)
- Intracellular, periplasmic, cell wall, or extracellular
21
Q
Affinity tag
A
- Used in affinity chromatography
- Recombinant proteins are typically expresed as a fusion with tag
- Include a protease cleavage site for removal
- Poly-his (His6) – Ni2+ resin
22
Q
Protein Purification in a nutshell
A
- Find or design method to detect protein (assay)
- Cell SourceAquire source (tissue, cells, etc)
- Cell disruptionLiberate protein from cells into buffer that enhances stability and solubility
- Debris removalRemove cellular debris and optimize yield + purity
- Initial purifcation heat denaturation/salts
- High resolution purification ex. ion exchange, size exclusion chromatography, affinity chromatography, electrophoresis
== Purified protein
23
Q
Claification after cell lysis
A
Removal of insoluable cellular material by centrifugation
(prevents columns from becoming clogged)
24
Q
Temp during purification
A
All steps should be done on ice
* Most proteins are more stable at 4°C than at room temp
* Proteases have a lower activity at colder temps
25
Precipitation
A good first step
* Most proteins are soluable at physiological salt conditions and neutral pH
* Salting in: Lower salt concentration = more soluable proteins (NaCl favors staying in solution)
* Salting out: Higher salt concentrations = proteins precipitate bc hydrophobic regions cluster together. (Ammonium Sulfate favors salting out)
26
Cations of Hofmeister series
N(CH3)3+ > NH4+ > K+ > Na+ > Li+ > Mg2+ > Ca2+ > Al 3+ > Guanadinium
- left = better at salting out
- Right = better at salting in
27
Anions of Hofmeister series
SO4 2- > HPO4 2- > CH3COO- > citrate > tartrate > F- > Cl- > NO3- > SCN -
- left = better at salting out
- Right = better at salting in
28
Salting out
* most effiencent at the pI of protein
* Hydrophobic attraction increases with temp
* Higher purity = higher protein solubility
* Never precipitates all protein
* Precipitation yields are best at high protein concentrations
29
Affinity Chromatography
Purification using affinity tags
1. Specific interation of the target with immobilized affinity ligand
2. Washing step where non-bound solutes are washed off
3. Elution step, where selectively bound molecules are eluted
- can be done with competiting substrate
- or by applying conditions that disrupt the specific interaction
30
Affinity Chromatography using His6 tag
Done with Ni2+ resin
* Attach his tag to recombinant protein
* Need to consider pH b/c protonated histidine is repelled by metal -- therefore more basic buffer solution (can also be used to elute)
* Use imidazole to elute his-tagged proteoms b/c imidazone competes woyj metal tag
31
Ion Exchange Chromatography - Concepts
* Separating proteins based on charge
* pH above pI = net negative charge
* pH below pI = net postive charge
* Protein has a positive charge = cation exchanger
* Protein has negative charge = anion exchanger
32
# Steps of:
Ion Exchange Chromatography - Basic principles
1. **Initial Stage** Beads have some counterions in buffer to maintain electric neutrality. -- Sample is loaded
2. **Absorption of target** ionic strength is adjusted so proteins bind to beads. Column is washed to remove uncharged molecules
3. **Starting elution** Salt gradient is used to elute bound protein. Low ionic strength elutes weakly bound proteins (w/ lesser charge)
4. **End of elution** Salt gradient increases. High ionic strength elutes tightly bound proteins (w/ higher charge)
5. **Column Regeneration** High ionic strength buffer washes through to remove all bound protein. Column is then conditioned to starting buffer
33
# ion exchange chromatography
Gradient elution vs. Step elution
Gradient elution - uses salt gradient
* helps you figure out what proteins elute at what concentration
Step elution - jumps in salt concentration
* you can get higher concentrations of target protein
Start with a gradient to figure out what concentration your target protein elutes at. Then use step to collect a highly concentrated sample
34
Size Exclusion Chromatography (SEC)
Seperating proteins based on size
* Small molecules enter spaces within beads = smaller molecules take longer to elute
* Large molecules cannot enter beads so takes quickest route = large molecules elute first
* Good polishing step to clean contaminent
* Can be used to estimate protein size
* Disordered proteins act as large proteins so elute quickly
35
# SEC
V0
SEC
Void volume
- volume outside beads
- the first volume at which any protein will elute
36
# SEC
Vt
SEC
Total volume of the column
37
Vt - V0
SEC
Separation range
38
Hydrophobic Interaction Chromatography (HIC)
* Uses same principle as ammonium salt precipitation
* reversible absorption of proteins to hydrophobic media/columns
* Load at high salt
* Elute by dropping salt concentration
39
What must we know to monitor purification
At every step we must know:
* the total protein concentration in each fraction
* The volume of each
* Target protein concentration in each fraction
The protein will be as pure as possible when ratio of target protein to total protein cannot be increased
40
A280
Used to calculate total protein concentration
* linear relationship between A280 and total protein concentration
* Approximately A=1 means 1mg/ml
41
Western Blotting
* Separate proteins by polyacrylamide gel electrophoresis
* transfer to a membrane
* detect protein using antibodies (one is labelled and can be visualized)
* Helps confirm protein identity
42
Mass Spectrometry
* Helps confirm protein identity
* Good for intact proteins with multiple charges
43
What can you use Mass Spec for?
* Intact mass measurement
* Sequencing proteins (every AA except lucine and isolucine have diff. masses)
* Post translational modifications (can find exact location)
44
Endopeptidases + Mass spec
Endopeptidase (proteolytic enzyme that cleaves a peptide bond within a protein)
* use it to cleave different regions of protein to make fragments
* Use mass spec to determine amino acid sequence
45
Problems with expression
* cleavage of target proteins by E. coli proteases. (need protease inhibitors or strains without certain proteases to block activity that can mess with protein expression)
* Aggregation of expressed proteins. Can be prevented through codon optimization, altered expression conditions, reengineering, or refolding techniques
46
Protein Ligand Interactions:
**Qualitative** Assays
**Show IF things bind**
pros
* faster
* high-throughput
cons
* more error prone
* false positives
Usually do qualitative first and then do quantitative
47
Protein Ligand Interactions:
**Quantitative** Assays
**Shows how tight things bind**
Pros
* more accurate
* less false positives
* detailed binding constants (telling you how tightly bound)
cons
* can be expensive
* slower
* requires more proteins
Do quantitative after qualitative to confirm binding and get more info
48
Differential Scanning Fluorimetry
AKA thermal shift assay
Heat up protein, add flourescent dye that has hydrophobic affinity. Dye binds to internal hydrophobic residues as protein denatures.
Measure the temp of unfolding with different concentrations of binding substance
* Melting point is proportional to how tightly bound a ligand is b/c ligand binding stabilizes protein
* Measuring melting point (Tm) shift between no ligand to high concentration of ligand
Pros:
* Inexpensive
* Fast
* Easy to run high-throughput
Cons:
* can easily fail
* Semi-quantitative
49
Equilibrium Dialysis
* requires an easy ligand assay
* requires proteins and ligands separated by membranes
Measure how much free ligand is in cell and use the known amount of ligand and receptor added to figure out how much bound ligand there is. Then use this formula to create a scatchard plot
Kd = [R][L]/[R * L]
B/F = Bmax/Kd - B/Kd
Slope = -B/Kd
x-intercept = Bmax/Kd (Stoich of the binding)
Pros
* inexpensive
* quantitative
Cons
* needs lots of material
50
Isothermal Calorimetry (ITC)
Adds a little bit of ligand to receptors. Binding releases bit of energy in form of heat. Then sample is heated and cooled back to baseline. Cycle is repeated until protein is saturated
Pros:
* low running cost
* VERY accurate - gold standard
* quantitative
Cons:
* expensive equipment
* needs a lot of material
51
Surface Plasmon Resonance (SPR)
Uses a chip with a bunch of receptors bound to it.
* Inject some ligand and binding will begin; the resonance signal increases == association period
* Sites start to become saturated and resonance signal flattens out
* Sites are fully saturated and ligand is no longer flowed in. Proteins will begin to dissociation. Speed of dissocation is proportional to how tightly bound
* The chip is regenerated through the addition of a solution which strips ligands off
kon = association curve
Koff = dissociation curve
Kd = koff/kon
Pros:
* very accurate
* extremely accurate and valuable data
* quantitative
Cons:
* expensive equipment
* high running costs
* can be tricky to set up
