Dialysis

Make sure to have 2 L of Dialysis (ITC) buffer prepared. You will need to place a stir plate inside the chromatography fridge and plug it in. You will also need a large beaker and a stir bar. Make sure the beaker is small enough to fit inside the chromatography fridge on top of the stir plate. Get a dialysis casset (make sure the MWCO is substantially larger than your protein's MW or else it will filter out of your casset). Hydrate your casset by placing it in water and waiting for the clear portion to look a little wrinkly. Once your casset is hydrated, grab a 5 ml syringe and a needle (It is suggested to use one of the longer needles). Rinse it with magic water and ITC buffer. Place about 800 ml of ITC buffer into your beaker. Once the syringe is rinsed, pull some protein into the syringe. The casset will have 2 places where you can insert the syringe (some have 4). Insert the syringe needle into the casset at a 45 degree angle. Pierce the membrane then pull the needle back so that it is around 1 mm inside the casset. Turn the casset over so that side with the syringe in it is parallel to the counter and begin slowly inserting the protein into the casset. Try to not make any bubbles when inserting. You can insert the protein in portions if you have more than what fits into the syringe. Once all your protein is inside the dialysis casset pull some air out of the casset so that the air pressure is not too high. Place the casset into the beaker with dialysis buffer and leave it stirring for 2-3 hrs at a relatively high speed (level 5-6) so that the liquid is moving throughout the beaker. Once 2-3 hrs have passed, switch buffers and let it spin for another 2-3 hrs. Once the last 2-3 hrs finish remove the protein from the casset using the syringe and place it in a falcon tube and leave it in the temperature needed. If you are planning on dialysing for ITC make sure to keep part of the last buffer.

ITC- loading

Make sure to program the ITC so that it cleans itself before beginning anything. Also make sure to rinse the syringe and cell with buffer solution so that any remaining solutions from when it cleaned itself does not effect the data. Make sure you centrifuge anything being used for the ITC to ensure all the solutions are well mixed before allowing the ITC to measure anything. To load the cell take the syringe and fill it to 300 ul and tap out any air bubbles. Then place the syringe into the cell and hit the bottom. Begin slowly (timing is key!) loading your sample. Once you reach 150 ul lift up the syring about half way and continue loading to reduce displacement. Once some liquid begins to gather at the top of the cell remove the syringe and try to suck up the liquid at the top. Some people recommend once hitting 150 ul to let out a few stronger and quicker pushes of the syringe to get rid of any bubbles, but we tapped the bottom of the cell which worked very well. Make sure to get rid of as many bubbles as possible since it will affect your results. You should end up with only 0-20 ul of sample left in your syringe. When preparing the solution that is going to be loaded make sure there are no bubbles in the test tube.

Make sure the cell and the syringe have reached room temperature before beginning equilibration. If they have not, your equilibration will take a long time and you will not get good data. Make sure to use a new ATP vial each run to lower the risk of recording the heat of hydrolysis. If you find that your data is very noisy and bumpy it could be due to a number of things. First, there could be a bubble in the cell in this case, you must redo the run. The concentration ratios of your enzyme to substrate to cofactor could be off or too high overall. Lastly, the contents in the syringe or cell have not reached temperature causing a bad equilibration and noisy data. If all of these could not be the case, you could also try reloading the reference cell or cleaning the ITC.

ITC (Kinetics) - How to analyze data

Open your raw ITC data into the MicroCal PEAQ-ITC software. We saved each of the ITC files as v vs S plots. We did this by making sure the enzyme markers are in the correct spots then clicking on presentation -> rate plot -> export data. We uploaded both raw ITC data and v vs S plots to this wiki. After getting all of our raw data we used Graphpad prism to begin subtracting controls and fitting our data into an S-shaped curve. We got a free trial that will end in about a month, but for future reference. After clicking on the icon to open the software you will select the X: Numbers and Y: Enter 2 replicate values in side-by-side subcolumns (If you must subtract a baseline) Enter and plot a single Y value for each point (If you don't need to subtract a baseline). Before putting any quantities into the Graphpad prism software, first open up the data set you are trying to fit into an S-shaped curve and the control you are subtracting in excel. Delete the first two columns of quantities (example title: DesDkineticsblankFit_X). You will then highlight the other two columns together (example title: DesDkineticsblank_X) and sort the numbers from smallest to largest. You must highlight both columns so that excell keeps the corresponding X and Y quantities together. IF YOU NEED TO SUBTRACT A BASELINE: You will then look at the X quantities of the data you are trying to fit into the S-shaped curve and the control. Try to find an almost same number in both (i.e. 0.01352 and 0.01332) towards the top of the data. You may delete anything above the number you chose to start your analysis with in both the X and Y columns. Now you will copy the X quantities of the data that you are trying to fit into a curve and place it into the X column (the one on the very right) of Graphpad prism. You will then copy the Y of the data corresponding with the X you just copied and paste that in the AY1 column. Then copy the Y data of the control and paste that in the BY1 column. You will then click the analyze button and select "Remove baseline and column math", make sure all data sets are being analyzed. You will then make sure the baseline is defined as Dats set B, click remove baseline(s) from the results, and Difference:Value - Baseline. If you do not need to subtract a baseline then skip those previous steps. Also make sure at the bottom the create a new graph of the results button is clicked. A graph will be made and it will probably Make an S and then drop down again. You will place your mouse over, you do not need to click, the point were the graph begins to drop again information about the point will appear look at which row that point is in. You will then go to the results table of that graph and highlight everything below that row and then go to Edit and hit exclude values or Ctrl. + E. It will then ask you if you would like to freeze these data points, hit yes. Go back to your graph and you will get a nice S- shaped graph. To put a line through this graph you will go back to your results table and above the Analyze button there are 3 buttons that look like a line graph hit the one in the middle (it looks like a classic enzyme kinetics graph and when placing your mouse over it it says: "Fit a curve with nonlinear regression"). You will then click on Enzyme kinetics - Subtrate vs. Velocity and either Allosteric sigmoidal or Michaelis-Menten. It will then fit your data into either a S-shaped curve or a classic enzyme kinetics curve, as well as, give you all the kinetics constants in another Results table. To find Kcat divide Vmax by the Enzyme concentration.

ITC- How to combine graphs

Click on one of the graphs you would like to combine. Then go to Add or remove data sets,... under the Change tab. Make sure you are in the Data sets on Graph tab and select the graph to be added then click Add on the right. You may then go to the appearance tab and create a color scheme for the different data sets. The magic wand icon under change at the top of the page makes the color scheme consistent throughout the notebook.

 

Nanodrop

Click on protein tab and select the protein A280 absorbance. Then for DesD samples, select the dropdown for "other protein E and MW". The E/1000 value for DesD is 90.760. The MW for DesD is 67.6385 kDa. Open the lever for the nanodrop and use a chem wipe with water to clean both ends. Follow the instructions that follow. First by loading the blank using the buffer that the protein was dialyzed in. Then load the sample. Each of these drops should be 2 uL. Refrain from getting any bubbles in the drop so you can get accurate readings. There will be a graph with peaks that pops up as well as three headers with their values below. The A280 is the absorbance of the protein, this is the primary focus. This should be below 1.00 and the target number is around 0.4. If it is high a dilution can be used to lower this number such as 1:10. The other value (A260/A280) shoudl always be around 0.75.