DSC ITC SPR Flashcards
What it DSC use for overall
What is it useful for
Used to study the physical properties of molecules and their thermal stability over a range of temperatures
Useful in finding protien stability (native protien stability, mutant, complexed)
Finding membrane phase state (gel, ripple, liquid crystalline)
What does DSC mainly use to find the thermal stability of a molecule
The change in Enthalpy (delta H)
Describe the DSC instrument
Describe how the instrument works
Sample chamber : contains The sample cell and reference cell
Power is applied to both cells to raise their temps at the same time, any changes in temp are due to the sample cell molecules changing
During this change heat is either absorbed or released, usually absorbed (endothermic) due to protien denaturation.
A thermocouple monitors the temperature difference between both cells and ensures the heat change between them is 0 (matches their temp) by adjusting the reference cell to match the sample cell temperature
What reactions usually aren’t seen with DSC
Exothermic bc applying heat to the system
In DSC How does the thermocouple adjust the reference cell to match sample cell in exothermic event
In endothermic
Exothermic: heat released, decrease in sample cell temp, reference temp decreases because thermocouple takes away power
Endothermic, heat absorbed by sample, higher temp in sample, reference cell temp increases because thermocouple supplies power
What is in the sample and reference cell for DSC
Ref: buffer
Sample: buffer + protien or lipid membranes
What is the output of a DSC experiment
The temp change (power supplied) of the reference needed to match the sample cell is proportional to the specific heat capacity (Cp) of the thermally induced process
So you get a thermogram of Cp (kcal/mol/deg Celsius) vs temp
The peak means the ref cell needed a change in power to match the sample cell, so higher heat capacity
How do you analyze the outputs of DSC
From the thermogram you can get : Tm, delta H, delta Cp (and t1/2)
Melting temp: Tm, temp found at max heat capacity (peak), corresponds to phase transitions like denaturation of protien or lipid phase change
Enthalpy of transition: delta H, area under the peak from T0 to T1 (start and end of peak)
Cooperativity of melting: T1/2, width of the peak at half max height, more width more complex mixture with diff protiens of system melting at diff temps, narrow more homogenous melting
What is ITC mainly used for
What does it usually measure
Used for the study of thermodynamic interactions: bio molecular interactions like protien ligand binding or protien membrane accosication
Usually exothermic reactions, heat released upon binding
Explain the ITC instrument and how data is collected
Isothermal: Not increasing/scanning across a temp range. Pick one temp for both sample and reference to be kept at constant temp
Syringe injects small volumes of ligand to sample cell solution (sample contains bio molecule of interest), binding causes thermodynamic exothermic response (-delta H)
Response is measured as the power needed to return sample cell to isothermal (initial) temp (because that the change in Enthalpy from the initial)
injections are repeated until binding saturation is reached (or not)
What are the ITC outputs
Raw data: Thermogram of heat release (microcal/sec) vs time shows heat response after injection of ligand
Treated data: inverse sigmoidal curve (kcal per mol ligand vs molar ratio ligand/protien)
Treated data gives K, delta S, delta H, stoich (N)
How do you analyze the ITC outputs
Treated data gives K, delta S, delta H, stoich (N)
Ka: slope
N: x value at inflection point , how many mol ligand bind to a single protien
Delta H: Y2-Y1 final minus initial response
What so important to note for ITC
If the binding is a change in entropy and not Enthalpy change then that interaction won’t be seen in ITC
Describe the main thing the SPR does
What can it tell us
Investigates real time analyte ligand binding kinetics and interactions, not based on thermodynamic response but instead optical activity (diff from ITC and DSC)
Tells protien protien , protien ligand, antibody antigen, drug binding (basically all binding events)
Most complex than itc and DSc
Describe the SPR instrument
A metal sheet (gold) coupled to glass slide has ligand bound by dexrtran on the Slide
Polarized light directed toward slide, upon total internal recollection (>99.99% of photons are reflected and not refracted) the elections from the gold delocalize and resonate parallel to the plate
These electrons resonating make a plasmons wave and make the plate sensitive to mass changes
Analyte solution passed over the plate, analyte binds, binding is detected by plasmons which change the chips refractive index: this changes the angle of reflected light
The change in angle of reflected light is measured as resonance units across the entire experiment (when ligand bound and when washed away)
What are the outputs of SPR
A sensor gram (RU vs time): gives k and K (rate and constants)
How do you analyze the output of SPR
Baseline
asscociation: Passing ligand over increases
RU, give ka
Plateau: all binding sites a saturated and no more ligand binding, give Kd
Dissociation: wash away with diff buffer to promote dissociation , give kd
Regeneration: washing away rest of ligand
Pros and cons of ITC DSC SPR
ITC:
Pros: stoich, Kd, delta H, delta S, label free, non destructive
Cons: noncovelent compexes show weak signals, slow takes time to optimize experimental setup (so we can get reproducible results), not suitable for rxn that take a long time, can’t detect entropy driven rxns
DSC:
Pros: non destructive, easy, give delta H, tm , t1/2, thermal stability
Cons: takes a long time to run DSC scan, in more complex mixtures it’s harder to determine which area corresponds to which lipid
SPR:
Pros: real time kinetics, label free, delta S/delta H independent, small sample size
Cons: needs uniform deposition into film, need ligand to be immobilized (can change conf and binding), non-specific binding
What does a small peak in DSc mean
Needs lower energy to phase change
Spr scaling of intensity with concentration
If the intensity of the spr curve does not scale with the concentration of ligand added, that means that at higher concentrations reaching full saturation at the surface of the chip
What needs to happen to the sample in DSC
Needs to be degassed
What is spr mainly used for
ITC
Drug binding
Energetics of binding events (because it gives delta h and S)
