Whether you are new to performing SPR experiments or not, everyone can use a brief refresher on Good Laboratory Practices (GLP). Using GLP will help ensure that you get the best data possible from your experimental design and make the best use of your valuable samples.
Tip 1: Spin down samples prior to dilution in running buffer. Spinning your samples in a centrifuge for just a minute or two will prevent problems later with clogged tubing.
Tip 2: Eliminate voids in tubing connections. Voids (or gaps anywhere in your lines) can lead to bubbles and poor peak shapes. To avoid this problem, make sure the tubing is seated properly. First, insert the tubing into the one piece fitting, leaving a 1/8” piece of tubing to seat against the port. Next, with one hand, push the tubing all the way into the port until it bottoms out. Use your other hand to seat the fitting against the port and screw in securely. PEEK ™, fittings can loosen over time, so always check that the fittings are tight at the start of a new day of experiments.
Tip 3: Eliminate air from all injections. Air in the lines can cause dropped data points and variable responses, and can sometimes lead to total loss of responses. The following steps can help eliminate air:
(a) Before using running buffer, thoroughly degas under a vacuum for about 10-15 minutes for buffers containing TWEEN® 20 and 25-30 minutes for buffers not containing surfactant.
(b) Use adequate volumes in vials and well plates so that your injector sample needle does not aspirate air.
(c) Slow down the Autosampler pickup rate for more viscous samples, e.g., those containing glycerol.
(d) For vials (or well plates that have caps), make sure the Headspace Pressure control feature is set to On.
(e) Perform full loop injections with 10% overfill. To reduce sample consumption, we recommend using a smaller loop rather than doing partial loop injections. Keep in mind that you can only inject about 80% of the loop volume before you start to see mixing, (you will see a decreasing signal in the association part of the injection). To determine if there will be a mixing problem, fill your conditions into the following equation:
Flow Rate (µL/min) X Injection Time (min)
Sample Loop Volume (µL)
The result should be 0.8 or less. If the figure is higher then you will need to reduce the flow rate, shorten the injection time or use a larger loop.
Tip 4: Minimize bulk shifts. Bulk shifts are non-binding related changes in refractive index that can interfere with peak shapes and your resulting data analysis. The following image contains data from an experiment that suffered from bulk shifts; as you can see, some concentrations have worse bulk shifts than others. The end result is that data are difficult to align, difficult to perform reference subtraction on and ultimately difficult to fit.
These steps will help you avoid any effects from bulk shifts:
(a) If possible, dissolve your samples directly in running buffer, or perform a buffer exchange into running buffer, using Zeba™ spin desalting columns or equivalent.
(b) If your samples are not directly soluble in running buffer, then match your sample solution to the running buffer as closely as possible. Keep in mind that only 1% to 5% DMSO in the running buffer is enough to keep most small molecules dissolved.
(c) Be sure to use running buffer in the Autosampler wash reservoir.
Please make sure you check-out Tip #1 in our Tips for Users Series with helpful information on how to identify and minimize the influence of mass transport
We hope you enjoy this column and return regularly for future posts, which will provide additional tips for your SPR experiments. You can read more about SPR elsewhere on Reichert’s website, including our first blog post The Resource.
We also ask you to provide your own input and suggestions to make this column even better. Contact us if you have any questions or topics you would like to discuss, or if you have certain tips of your own that you would like to share.