Isothermal Titration Calorimetry
Buffers have been found to need a high buffer capacity to control ATP and citrate pHs, as well as 5 mM reducing agent and 25% glycerol for stability. Recipes can be found here: recipes#ITC_Buffer?
For DesD, use 20 uM enzyme in the cell compartment, and keep all substrates (0.20 mM) together in the syringe.
Run 15 injections over 30 minutes.
___ uL/ injection
On a Malvern PEAQ ITC, the following conditions worked for FslA
- In the cell, load 300 uL of 5 uM or 10 uM enzyme (use 5 uM if the heat signal is sufficient). No bubbles, no troubles.
- In the syringe, load 5 mM each of ATP, putrescine (amine substrate), & citrate, all brought up in dialysis buffer from powder, if possible into 20 mM stocks. (note, pH citrate to 9.0 before using or risk ridiculous background heat)
- Run single injection method kinetics with the following parameters:
- 37oC temperature
- 30 uL injection over 180 seconds.
- reference power at 30 ucal/s
- stirring speed at
- watch for 30-60 minutes
Run appropriate controls (substrate into buffer, everything but the amine substrate into enzyme, heat inactivate the enzyme) but analyze in GraphPad Prism or other non-proprietary software as the PEAQ kinetics software can't handle cooperativity.
This assay allows a variety of carboxylates to be examined for flexibility/specificity in primary substrate. The hydroxylamine, when in a peptide bond, forms hydroxamate. Hydroxamate will absorb wavelengths at 540 nm if complexed to iron ions. It's important to note that this assay detects maximum activity over 30 minutes, and is not an initial rate assay.
Excel Spreadsheet for calculations:
Kinetics Assay Cacls.xlsx
NIS assay protocol
- Assemble the assay reaction to the following final concentration:
30mM Citric Acid, a-ketoglutarate, or other carboxylate
200mM NH2OH HCl
100mM Tris-HCl pH 8.0
2-3 uM NIS synthetase
18ohm distilled water to 500 uL
- incubate the reaction mixture for 30 min. at 37C/310K
- prepare 500 uL of stopping solution:
3.3% Trichloroacitic acid
10% (w/v) FeCl3
18ohm water to a final volume of 500 uL
- after the 30 minute incubation period add 500 uL of stopping solution to the reaction mixture to unfold the protein and complex the hydroxamate product with the ferric iron. let this sit for a few minutes.
- centrifuge the solution to remove unfolded protein, and measure absorbance at 540 nm using Varian Cary 50 Bio UV-Vis Spectrophotometer in doctor Watson's lab.
- For the blank, add everything to the mixture but the protein. Use 18ohm water in place of the protein volume.
- When taking absorbance of the blank, do not actually blank the machine. Take the absorbance of the blank and all trials of the assay, and then substract the blank from the trials yourself.
- If you are getting negative numbers, could be indicative of a non-functional protein, cuvette not placed correctly in the spec, or a mistake was made in the assay itself. Re-do the assay and see if you get the same numbers.
- Always a good idea to do the assay in triplicate.
Use above reaction concentrations, but omit ATP and MgCl2 entirely. For the carboxylate, use 3 mM to start, and Coenzyme A (negative control), succinyl-CoA (substrate) or some other acyl-CoA (to test for flexibility in recognition). The rest of the protocol should be the same.
UV-Vis absorbance reading
For any of the BioMate UV/Vis machines, prepare separate blanks and reaction cuvettes. Set the variable read wavelengths program to 540nm, and read as many samples as you have cuvettes, recording the numbers as you go.
The Varion kinetics machine
- Turn on the water bath and dry bath and bring to 37 degrees C. Turn on the UV/VIS and computer, if not already on. Wait for the machines to complete any calibrations (i.e. stop making noise) before opening any software programs.
- For this assay, either the kinetics or the simple reads program will read absorbance at 540 nM. For Simple Reads (from either the desktop shortcut or the start menu):
- Click connect to get the computer and UV/Vis communicating
- adjust settings/set-up to wavelength: 540nm (if in the kinetics program, also set data collection: at the end, and make sure autosave is on.)
- Use the appropriate height adjustment for the cuvette and reaction volume: if you have a 1 mL reaction, the meniscus can scatter light if the cuvette is used without height adjustment; a 2 mm cap for a quartz cuvette will provide an appropriate boost to center the beam in the reaction. If you are using a 0.5 mL reaction, the Watson lab has Frankenstein'd a modification to the cuvette holder with a bit of tape tabbed out of the top to center the beam appropriately. If you are getting nonsense results, you may want to be sure the cuvette is positioned appropriately.
- press the zero to blank (only takes few seconds). You may want to blank with just the Tris-HCL pH 8.0 to begin with, but make sure you run a negative control to determine your background absorbance, if you do. In the kinetics program, you may be prompted to add file name, it would be smart to save the file in the Hoffmann folder as well at that time.
- replace the blank with reaction sample,
- click okay button
- record results immediately in another program, as you go. (For the kinetics program results, you can adjust the range to 0.1-2' and recalculate if appropriate. Take the initial rate measurements from the slope and copy into a graphing software program as you go.)
- results: Abs = 0.1-1AU to be in optimum range of the detector.
Turn on the N2 cooling tank, which will take about 20 minutes to warm up.
Ensure that the N2 float is above 20 on the front of the CD
Then turn on (in order):
-the computer (if needed)
-the Jasco Circular Dichroism Spectrometer
-The water bath (there are two switches: the temperature control switch and the circulation switch)
-the table-top Peltier
Double check that the Julabo water bath screen shows it's on, if not, press ok.
Run the Jasco Spectra Program
From the desktop, double click the Jasco Spectra Icon
In the main window, choose spectra measurement from the left-side menu
A new window will pop up, with the new pull-down menu at the top active: select