FAQ - Questions around Microaraying
Both types of slide have intrinsically low non-specific binding properties. Therefore, blocking might not be necessary if the resulting increase in background can be tolerated. How much the background increases will depend on the nature of the target solution and the conditions used. Reducing the probe concentration to avoid comet taíl formation will help to reduce the need for the blocking step.
The preferred option is to store the slides directly after printing and immobilization steps, but before the washing and blocking steps. A safe and convenient way to store slides is to re-use the orginal slide boxes. If the slides are stored properly (dry and dark in a desiccator, ideally under an inert atmosphere) the background increase should be minimal. The printed slides are stable for several months.
If the slides have to be stored after the washing and blocking steps, the hybridization should be carried out within a few days. The reason is that the background flourescense of the blocked slides will greatly increase in a short period of time.
Comet tails are formed when unbound nucleic acids bind to the activated slide surface around the spot during the slide processing steps i.e. during the washing steps before blocking.
Several users have reported that fast and vigorous shaking during the first washing/blocking steps also helps to reduce comet tail formation.
Aminosilane coated slides
After spotting Aminosilane coated slides, such as NEXTERION® Slide AStar and A+, it is important to covalently immobilize the bound molecules by baking or UV-crosslinking and to inactivate the slide surface by blocking to prevent re-binding. Additionally lowering the concentration of nucleic acids in the spotting solution will help to reduce the excess of unbound molecules.
All other NEXTERION® coated slides
For active slide chemistries like Slide E, AL, P and H, the best approach would be to wash slides directly after immobilization with a specific chemical blocking solution. This will help to block/inactivate the surface immediately and prevent excess probe material from binding to the surface. Additionally lowering the concentration of nucleic acids in the spotting solution will help to reduce the excess of unbound molecules.
Block E should be diluted 1:4 with double distilled water and concentrated hydrochloric acid (HCl 37%) as shown below:
- 100 ml NEXTERION® Block E + 300 ml double-distilled water. + 80 μl of HCl (HCl 37%)
- 500 ml NEXTERION® Block E + 1500 ml double-distilled water + 400 μl HCl (HCl 37%)
- 1000 ml NEXTERION® Block E + 3000 ml double-distilled water + 800 μl HCl (HCl 37%)
- Undiluted NEXTERION® Block E pH = 5.0
- Diluted NEXTERION® Block E pH = 2.0
The diluted NEXTERION® Block E solution is stable for several weeks at room temperature.
The chemical deactivation is not recommended for protein applications as the deactivation solution can damage the bound proteins, or might have unpredictable effects on them. Chemically de-activating (or inactivating) the Aldehyde surface prevents non-specific binding of the target molecules (unless they are very sticky). In the protein protocol, the reactive Aldehyde groups on the surface are not chemically deactivated, but "occupied" by "blocking molecules" (e.g. BSA) this normally has no adverse effect on further reaction/incubation steps. On the blocked surface target molecules are prevented from binding, so the blocking step has the same effect as the chemical deactivation.