By Ashok Chavan, Ph.D. Photoaffinity labeling is one of the most effective techniques used by life science researchers to study protein/nucleotide interactions. Its popularity stems from the relatively easy experimental setup and the wealth of useful information that it can provide. The most common chemical modification of natural ligands for this purpose is to introduce an Azido moiety as the photoactivatable group. This group is either attached as a phenyl azido functionality or the modification is directly introduced into the parent structure. The phenyl azido functionality can be attached through either the phosphate or ribose moiety. Compounds such as 8-AzidoATP and 8-AzidoGTP are examples of azido group introduced into the parent structure i.e. base ring. GTP[g]-4-Azidoanilide shown below is an example of the phenyl azido functionality attached to the g-phosphate. Both classes of compounds have been used to study G-proteins.
Structure of Guanosine 5'-triphosphate [g] 4-Azidoanilide, (GTPgAA) ALT, Inc. maintains a modern synthesis and quality control facility located in Lexington, Kentucky. The facility is dedicated to the research and development of these compounds. This commitment allows us to guarantee the highest quality and to focus on new products. Full technical support is provided by personnel experienced in photoaffinity labeling. This brochure is third in a series of technical articles provided as a service to our customers. Proper Handling Instructions: 1. Removal of methanol: Probes are shipped in absolute methanol. With the required amount of the probe aliquoted, methanol can be easily removed by evaporation in a vacuum concentrator or under a stream of nitrogen. The dried probe is now ready to be dissolved in an appropriate buffer. 2. Recommended working environment is ordinary room lighting. Normal room lighting has not been found to significantly activate probes. However, protect probes from inadvertent exposure to UV light. 3. Safety Precautions: Use acrylic shielding (minimum 3/8" thick), as required, to reduce radiation exposure due to 32P. Essential Tools For a Photoaffinity Labeling experiment Protein samples mixed with photoprobe up to a final volume of 100 µl can be photolabeled effectively in a micro-tube (1.5 µl) using a hand-held UV lamp. For larger volumes (up to 200 µl), a 2 ml flat bottom tube can be used. For photolabeling in volumes >1 ml disposable plastic weighing boats have been found to be effective by providing smaller path length for activating light. UV source: Azide substituted nucleotide probes are photolyzed within 1-2 min with a hand-held UV lamp (254 nm). These lamps normally have intensities in the range of 4-6 mW/cm2 which is sufficient for good photoincorporation. The photochemical properties of azido-nucleotides are different than natural nucleotides which require 30-60 minutes irradiation at 2000-3000 mW/cm2 to afford direct photochemical cross-linking. Such high energy irradiations are usually damaging to most proteins. For the benzophenone and acetophenone substituted GTP photoprobes described in this brochure, the photoactivation requires UV irradiations >300 nm and can be achieved in two different ways. One method is to use hand-held UV lamp (254 nm, 3-4 mW/cm2). The intensity of the lamp should be checked with a light meter. If the intensity is lower then removing the face filter is recommended to achieve the desired intensity. Usually 2-10 min irradiation affords efficient photoincorporation.. Anther method is to use hand-held UV lamp at l 365 nm for extended periods, 1-2 hr, (Model E-16, Spectroline, Spectronics Corp. NY with an external Corning C 7368 plane glass filter, Hickey Corporation, NY to eliminate radiation l 340 nm). The lamp is usually held at a distance of 4 cm from the surface of solution. In both the cases the optimum time required for photolysis should be determined with preliminary experiments. Selection of Proper Photoprobe ALT, Inc. offers [g32P]GTPgAA with two different specific activities.
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