SPR Experimental Procedure
1 gold surface on glass slide
50 mL 0.5 mM alkanethiol solution
1 1.5 mL aliquot EDC solution
1 1.5 mL aliquot NHS solution
5 mL 8M ethanolamine solution
0.75 mL 1 micromolar Protein A solution
0.75 mL 0.2 micromolar cadherin solution
0.75 mL BSA solution
1 L buffer solution
1 L of DI water
3 disposable 5 mL syringes
3 disposable 1 mL syringes
SPR sample cartridge
Large glass bowl
50 mL glassware with valve
500 mL beaker for collection
1) Add 2.28 g EDC to 10 mL DI water, dissolve (152 mg/mL)
2) Fill near 15 mL, adjust pH to 7.5
3) Add in small volume to reach 15 mL
4) Aliquot solution into 10, 1.5 mL tubes and store in -20 freezer.
1) Add 0.222 g NHS to 10 mL DI water, dissolve (14.8 mg/mL)
2) Fill near 15 mL, adjust pH to 7.5
3) Add in small volume to reach 15 mL
4) Aliquot solution into 10, 1.5 mL tubes and store in -20 freezer
1) Dissolve 7.32 g ethanolamine in 12 mL DI water (1.464 g/mL)
2) Adjust pH to 7.5, and add water to obtain total volume 5 mL.
3) Aliquot solution into 10, 1.5 mL tubes and store in -20 freezer
Composition: 150 mM NaCl, 20 mM HEPES, 2 mM CaCl2
1) Dissolve 8.766 g NaCl, 4.766 g HEPES, and 0.294 g CaCl2 dihydrate in 950 mL of water
2) Adjust pH to 7.5, and add water to obtain total volume 1 L.
Protein A solution
1) Obtain Protein A stock solution from -80 freezer.
2) By taking some of the Protein A stock solution and diluting it with buffer, create a 0.75 mL of a 1 micromolar Protein A solution.
1) Obtain stock solution of cadherin of interest in HEPES buffer.
2) Using the stock solution and the buffer, create 0.75 mL of a 0.1 micromolar cadherin solution.
1) Incubate gold slide in alkanethiol solution overnight. Minimum time to form dense packed layer is 11 hours.
2) Prepare all solutions you will need for the experiment. The protein solutions should strictly be made the day of the experiment, and should only be stored at 4°C for a short time (2-4 hours) before use.
3) Disassemble the sample cassette, remove the previous gold slide if it is still in the sample holder, and rinse the cassette with DI water, cleaning it thoroughly, and then dry it.
4) Fill the large glass bowl with DI water, and submerge the pieces of the sample cassette.
5) Use a 5 mL syringe to fill the supply lines of the cassette with water, and then use the syringe to remove any air bubbles that may be attached to pieces of the sample cassette.
6) Remove the gold slide from the alkanethiol solution. Rinse the slide with ethanol, and then with water. Submerge the slide in the large glass bowl. If any air bubbles are present on the gold surface, remove them using the syringe.
7) Ensure that the o-ring is properly seated in the Teflon piece of the sample cassette.
8) Place the glass slide, gold surface up, on the metal piece of the cassette. Place the Teflon piece with O-ring on top of the gold slide.
9) Tighten the fasteners on the cassette until they are just finger tight. Over-tightening the fasteners can crack the gold surface and ruin the experiment, so care must be made. Simultaneously, if the system is not water tight, air will leak in and also ruin the experiment. So the right level of tightening must be achieved. This can be practiced assembling the cell with a glass slide that is not coated with gold.
10) After the fasteners are tight, remove the sample cassette from the water. Check to ensure the fasteners are still finger tight, adjusting them if necessary. Dry the outside of the cassette with a Kimwipe.
11) Wipe the SPR prism clean of any residue from the previous experiment. Apply the optical oil to the top of the prism, just a drop or two. Place the cassette on the prism, ensuring it is seated properly, and slide it around a bit to spread the oil and obtain good optical contact. When the optical contact is good, attempting to gently lift the cassette off of the prism shouldn’t succeed.
12) Once the cassette is placed, turn on the laser and the motor. Look at the sensor, and make sure the laser is striking the sensor.
13) Open the SPR software, and the Data Box programs.
14) Press the button where you are measuring as a function of angle. Note the shape of the curve. It should be smooth, and concave up. Ideally, the minimum should be positioned at an angle measurement over 20,000.
15) Save this water background, and export it as text.
16) Press the button where you are measuring as a function of time. An initial data point shall be taken, and another data point will be taken every 40 or 60 seconds.
17) After 5 minutes, pour some buffer into the glassware with the valve, and hook it up to one of the flow tubes through the sample cassette. Ensure when you do this that there are no air bubbles trapped in the line. Open the exit valve, and open the inlet valve so that you achieve very slow flow. It should take 5-10 seconds for each drop to form.
18) Allow the buffer to fill the system. This may take 20-30 minutes.
19) When the angle of the minimum has stabilized, retrieve 1 1.5 mL tube of the EDC and the NHS solutions from the -20 freezer.
20) Using a 5 mL syringe and a needle, draw up both the EDC and the NHS solutions into the same syringe. Agitate and mix by pushing 1 mL back into one of the tubes and then drawing it up again 5-7 times. After that, remove all air bubbles from the tip of the syringe.
21) Stop the buffer flow, and close the flow valves. Attach the syringe containing the EDC/NHS mixture to the inlet tube. Inject the EDC/NHS mixture over the course of 2-3 minutes. You should see an immediate large shift in the location in the medium. This is solely due to the index of refraction change. It will occur several times during the experiment. Do not panic.
22) Close the flow valves and allow the EDC/NHS to react with the surface for 15 minutes. At the end of that time, retrieve the Protein A solution from 4°C storage.
23) Draw up the 0.75 mL of Protein A solution into a 1 mL syringe. Ensuring that no air bubbles enter the inlet flow tube, attach the 1 mL syringe to the inlet. Slowly inject the solution. The injection should take 3-5 minutes.
24) Let the system stand for 15 minutes while the reaction occurs. At the end of that time, retrieve a 1.5 mL tube of 8 M ethanolamine from the -20 freezer.
25) Draw the ethanolamine solution into a 5 mL syringe, and then attach the syringe to the inlet flow tube. As always, ensure no air bubbles are introduced to the system. Inject the 1.5 mL of ethanolamine solution over the course of 2-3 minutes, and then let the system sit for 15 minutes.
26) After 15 minutes has elapsed, begin slow flow through of buffer again as you did in step 17. Allow the buffer to flow through for 30 minutes to re-establish equilibrium in the system. At the end of that time, retrieve the first sample protein, either cadherin or BSA, from 4°C storage.
27) Draw 0.75 mL of sample protein solution into a 1 mL syringe, and slowly inject it into the system. Again, ensure that you do not introduce air bubbles into the inlet flow tube. Let the system sit for 30 minutes with the sample protein solution.
28) Resume slow flow of buffer over the system for 15-20 minutes to wash off any sample protein cadherin that is not bound to the surface. At the end of that time, retrieve the second sample protein from 4°C storage.
29) Repeat step 27 for the second sample protein, and then let the system sit for 15-20 minutes.
30) Resume slow flow of buffer, allow flow to go for 15-30 minutes.
31) Close all of the flow valves, and stop the data collection software.
32) Save the data files, and export the file that plots the minimum versus time as text.
33) Shut off the laser and the motor and close the software.
Data Analysis Protocol
1) Use the Microsoft Excel template to convert the angles from goniometer units into degrees.
2) Note the angle in degrees of each minimum, and save that data to an excel file.
3) Open the software program supplied by the Corn Group.
4) Depending on your experiment, choose either a 4 layer or 5 layer system.
5) Input the given parameters, and make an estimate of layer thickness.
6) Based on the estimate of layer thickness, determine the effective index of refraction.
7) From the effective index of refraction, find the volume fraction of protein versus water in the layer.
8) From the volume fraction, determine density and effective coverage.
SPR Maintenance and Troubleshooting Guide
What maintenance tasks have to be performed on the SPR?
If the SPR is being operated properly, it requires relatively little maintenance. Keeping the area clean, making sure people wash their glassware, and occasionally wiping the prism with ethanol if it looks dirty. Most problems with SPR data originate from poor optical contact between the sample and the prism, or with problems in the sample, not with the instrument itself.
What can go wrong with the SPR?
The primary thing that can go wrong with the SPR is something changes the optical alignment. When that happens, the intensity/angle curves go from the smooth, parabolic type shape that is proper, to almost any other shape, square wave, flat line, sinusoidal, depending on the problem. When someone claims that the SPR is broken because of the curve shape, remove the cell from the prism, clean the bottom glass surface of the cell and the top glass surface of the prism, reapply the oil and try again. That will test the optical contact. When you have good optical contact, it will feel difficult, but not impossible, to lift the cell off of the prism. Second, the gold slides you are working with must be clean and high quality. How long ago did you deposit? Did you let them sit in air? Is the evaporator functioning properly? Many irregularities in the SPR curves can be traced to less than optimal gold surfaces. So, if you’ve checked the optical contact, make sure the gold surface is one that you trust to be high quality. If you have good optical contact and you are confident in the gold surface, your problem is with the optical alignment.
What are the five minute fixes to optical alignment?
First of all, check the angle reading for the prism on the goniometer, make sure the angle is set to ~22.5°. If not, set it to that position, and try again. Second, if the system still isn’t generating good results, set the prism to 0° on the goniometer, and adjust the laser to ensure that the reflection off the prism is shining directly back at the source. Then reset the goniometer to 22.5° and try again. These two changes will solve most of the times you are informed that the SPR is out of alignment. You should not need to adjust the laser more often than once every 18-24 months unless there is something mechanically wrong with the equipment.
Okay, I tried those, and the SPR still isn’t working. What now?
If that doesn’t solve the problem, you’re going to have to figure out exactly where in the optical beam you believe the disruption is. To check the laser path, fold a corner of a Kimwipe into a point, and put it in the path of the laser so it shines red. Then move the Kimwipe, and trace the path. Ensure that the beam is striking the prism, and that the beam leaving the prism is striking the detector. If that looks reasonably normal, check the level of the prism. If you put a level on the prism, you should find that the bubble moves to the center of the level when the goniometer reads ~15°. Also, if you notice that the path of where the laser strikes the detector is showing variance wider than the detector, you may have to adjust the prism position such that the spot where the laser shines on the gold slide is on or close to the axis of rotation for the goniometer. Once the prism is positioned, it should not be adjusted. Generally, the prism will only need to be reset if an SPR goes years without being used or if the equipment is moved.