SPR theory is not always easy to practise. Often your experimental conditions are different than the published or your system behaves differently than you expected. There are some general procedures which you can follow to speed up your experiments and get your data more easily. In this section there are some 'HowTo' questions to help with your experiments.
How to make a reference cell?
A reference cell can be used to compensate for matrix effects, refractive index effects and non-specific binding of the analyte. It is important to match the reference cell as close as possible with the other cells. Just activation and deactivation is not the ideal control. Try to use an inactive ligand or a similar protein like a non-related IgG or use BSA to mimic a protein surface.
Non-specific binding can occur between the analyte and the dextran-matrix or the ligand. The source can be the analyte or a contaminant in the sample. Matrix related non-specific binding can be overcome by using a different type of sensor chip. In addition, a different immobilization chemistry or blocking solution may be used.
For further reading look at the immobilization section.
How to get a stable baseline?
Drift in the signal comes from many sources. The instrument itself with all the electrical and optical circuits has its own drift. The manufacturer has put a lot of effort in minimizing this kind of drift but you cannot change the instrument characteristics. Other sources of potential baseline drift are temperature fluctuations, flow rate changes, air bubbles, regeneration solutions and sample dispersion.
Thoroughly degassed buffers will help to minimize the effect. When using a method, a high flow rate (e.g. 100 μL/min can be used to flush out small air bubbles between cycles. In addition, sufficient equilibration after immobilization is necessary because of the chemicals used. It is recommended to equilibrate the system over night after immobilization. In addition, when running a method, add a 5 minute wait time before the first injection to get a stable baseline at the start of a sensorgram.
How to check for mass transport?
Binding of analyte (A) to the ligand (L) on the sensor chip can be described as a two-step event. First, the analyte is transferred out of the bulk solution towards the sensor chip surface. Second, the binding of the analyte to the ligand takes place. The first step is also known as mass transfer and is carried out by convection and diffusion. Both events have their own rate constants (km and ka/kd). The coefficient for mass transfer (km) is the same in both directions. With full or partial mass transfer, the diffusion from the bulk to the surface is slower than the rate of binding of the analyte to the ligand creating a shortage of analyte at the surface.
Mass transfer limitation can be checked by using different flow rates. When the association and dissociation rate constants are independent of the flow rate, no mass transfer limitation is to be expected (read more at the Experiment page).
In order to reuse the sensor chip surface the analyte must be removed, but the ligand must stay intact. This so called regeneration procedure has to be evaluated empirically because the combination of physical forces responsible for the binding are often unknown, and the regeneration conditions must not cause irreversible damage to the ligand.
The most frequent method used, is to inject a low pH-buffer such as 10 mM Glycine pH 2.5. This works probably because most proteins become partly unfolded and positively charged at low pH. The protein binding sites will repel each other and the unfolding will bring the molecules further apart.
Other procedures use high pH, high salt or specific chemicals to break the interaction. It is important to choose the mildest regeneration conditions that completely dissociate the complex.
Therefore, no single regeneration solution can be recommended. For more information go to the regeneration page.
Once in a while you are done with experiments on a particular sensor chip, but the surface is still performing well. So you want to store the sensor chip and reuse it another time.
There are two options to store the sensor chip:
- Wet in a solution compatible with the sensor chip and ligand (4 °C)
- Dry, desiccated in a nitrogen environment (4 °C)
Which of the two is the best, you must find out yourself.
Most important is that the surface is as clean as possible. Be sure to remove all the bound analyte or captured proteins and lipids before removing the sensor chip from the machine. The storage buffer used can be the running buffer but adding an anti-bacterial agent is recommended (make sure it is compatible with the ligand).
The accurate protein concentration is important when fitting the curves to extract kinetic parameters. Even with affinity ranking, proper concentration estimations are necessary. The 'How to measure protein concentration' document will list some of the possible protein determination possibilities.