Fluorescense Applications Flashcards
What is accosiaton vs dissociation equilibrium
What is Kd if 2 binding sites on protien
What if equal binding to all sites binding site
Ka: P+L = PL and pl/p + L
KD is opposite
KD1 : P x L /PL, Kd2 = PL x L / PL2 (product over reactants in the equation)
KD1=KD2=KD3
What are spectroscopic titrations used for
Measuring the binding of one molecule (usually a small molecule) to a second molecule (usually a macromolecule)
What are the steps to spectroscopic titrations
- Place high [P]tot in both sample and blank cuvettes
- Measure the spectroscopic parameter: either abs, fluoresce, change in abs from CD, etc
- Add a small amount of ligand to the sample cuvette, measure response
then repeat many times
What are the outputs for spectroscopic titrations
Tight binding
Moderate binding
Describe the plot when there is tight binding
What does it always start at
Plot: Change in spectroscopic response (ex change in fluor) vs (L] tot (ligand concentration that we titrated in)
Since looking a change in response, the response (y) always start at zero (sample and ref intially have zero change in response since same)
as ligand bind change in response increases rapidly, sharp transition to a straight line since all binding site occupied (binding has stopped)
Describe the ways N can be found in tight binding
N= number of ligand binding sites for the macromolecule
[L]sat’d = N [P]tot
So the concentration of ligand at the saturation point (when is start to get straight line) equals the stoic times the total protien concentration
Rearrange to get N
- Calculate delta e1 and delta e2 then N=deltae1/e2
delta e2 is the First diagonal line of curve (intial binding) delta e1 is the second straight line part of the curve (at saturation)
Step 1: finding delta e1 per macromolecule (change in response due to protien)
Chang in delta e1 =detaATot/P tot
Delta A total is from A0 to A sat’d
Step 2: finding delta e2 per ligand (change in response due to ligand)
Slope= delta e2 per ligand= delta A sat’d / L sat’d
A0 to A sat’d/ concentration of ligand at saturation
Step 3:
N= delta e1 / delta e2
So N is proportional to concentration of P and L
Explain how to find Kd for moderate binding (more curved)
You need to have the P tot < 10 times the KD so that the concentrations of P and L aren’t too small
If ptot is too big, it’s tight binding not moderate, so no curve, concentration of L=0 (since little amount of binding happening), this makes KD wrong
Explain why we have to do a second experiment for weak binding cases to find N
Whag is the second experiment, what special
Since in weak binding there’s more curve and the slope is changing through, the delta e2 from the intial slope isn’t accurate , can still find delta e 1
Second experiment: main goal is to get the slope for delta e2
Simulate very tight binding early on: use very high p tot so that there is tight binding at lower concentration of L tot
This give linear slope to find delta e2
Don’t need to go to full saturation because we already know delta e2 from the moderate binding plot
Whag is non linear fitting
Can use a computer model to make a non linear fit of the titration curve .
It finds What value of KD and delta A sat’d (change in Spectroscopic response at saturation) give best curve
This curve gives the best fit and best KD, but it uses assumptions to do so
May be assuming N=1, need to make sure it actually does equal 1 because curve looks diff if more than 1 binding site
How do we use indicators in spectroscopic titrations
Method:
Add P total and D (dye) total to solution
Add a small amount of calcium
Measure the delta A or delta flour after each addition of ca
Theory:
The indicator dye changes colour when bound to the ligand which is usually calcium
P + ca makes PCa
Same for D
So find Kd for dye binding to calcium and that’s the same for the routine binding to calcium
Explain the range of diff types of spectrophotometers
Classic large ones: have settings to optimize, can have well plates to have sample in many diff wells to do many Fluor assays at once (like cell death assays)
Small ones: handheld device to measure Fluor of anything on the go
What is total internal reflection fluoresce
Like spr, needs total internal reflection
When this happens, an Evanescent wave is made in the sample which propogates the excitation of fluorophore S in a very small slice of the sample (100nm)
In comparison to a cuvette, exciting each fluorophore across a 1cM path length (may miss some fluorophore), but for this every one is excited whcih gives clear fluorescence signal
This can be used in image of stained cells because it give high quality images
Explain the role of each cuvette in Fluor
Four sided plastic cuvettes : used for flour because measuring light at 90 deg
Not four sided: measure abs because measuring straight across (180 deg)
Cuvettes with small optical window: used for solutions with high sensitivity, the smaller window Minimizes the intensity, has 10mm path length
Flow through cuvette: has one end connected to a syringe that passes sample through the cuvette. Measures the changes of flour over time/volume of solution. So not just static Fluor but dynamic
Plastic: bad for low wavelengths, start to absorbs light at 350 and below
Quartz: better for low wavelength (far and near UV so used in CD), gives more sensitive readings because limit refraction of light
Optical window at bottom: good for samples where there is aggregation and sedimentation
Triangular cuvette: good for turbid samples if can’t fix them , decreases path length without having to use small volume cuvette, exciting in a smaller window.
Why is it better to use small volume cuvette (shorter path length) rather than large volume
Low volume of sample reducing the amount that light need to travel in a turbid solution (reducing inner filter effect)
Better if don’t have enough sample (can use as low as 15 microlitre) usually need 2.5mL for 1cM pathlength
If less sample , Want to avoid hitting the meniscus with the excitation light, lead to less emmision intensity because light passing though and exciting a little bit but not much
What are multichannel pipettes
Fill many well at the same time, takes less time
