Tips for Users Series - #3 - Get better data with Good Laboratory Practices (GLP) - Part 2
Part 2: Our last blog post laid out four tips to help ensure you get the best data possible from your experimental design. This post contains four more tips related to Good Laboratory Practices (GLP):
Tip 5: Eliminate carryover. You should always do an adequate number of washes to completely remove all traces of the sample and regeneration solution from the injector and sample loop. Usually one or two washes is adequate, but more washes may be needed for sticky samples or if concentrated regeneration solutions are used.
In this example, 6M guanidine HCl is used as the regeneration solution. Because not enough washes were done after the regeneration solution was injected to wash the regeneration solution from the injector and sample loop, the second sample injection (and all subsequent sample injections) showed no response:
Performing the following steps will help make sure you do not have carryover problems:
(a) Fill the wash bottle with your running buffer – be sure to use a large enough volume of running buffer in the wash bottle to do all the washes throughout your run.
(b) Run blanks - if you see a response in the blank it is an indication you may need to program more washes.
Tip 6: Serially dilute your samples. This will help minimize pipetting errors. We recommend regular dilutions of 1:2 or 1:3.
Tip 7: Always run replicates, blanks and at least four or five sample concentrations. We recommend re-injection of samples two or three times. This shows that the sample responses are not degrading over time and also ensures that the regeneration solution (if needed) does not denature the immobilized protein. Blank injections can help correct for small shifts in baseline over time. Injection of multiple sample concentrations makes the kinetic fits more reliable. As the following figure shows, the best results are obtained when replicates, blanks and injection of at least four or five different sample concentrations are done.
Tip 8: Repeat the experiment on multiple days. By comparing results from repeat experiments, you will increase your confidence in the quality and trustworthiness of the data. For small molecules that can reach equilibrium, the KD values obtained from kinetic data and form plots of the Langmuir isotherm also will agree.
As an example, carbonic anhydrase is immobilized on a dextran chip via amine coupling. 4-Carboxybenzenesulfonamide is then injected over the surface at different concentrations. Since responses reach equilibrium, the kinetic data and Langmuir isotherm data agree.
And the data compared from 4 different days agrees.
We hope you enjoy this column and return regularly for future posts, which will provide additional tips for your Surface Plasmon Resonance experiments. You can read more about Surface Plasmon Resonance elsewhere on Reichert’s website, including our first blog post and our first tip on how to identify and minimize the influence of mass transport.
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.