The Basic Set Up for Photolabeling
The Basic Photolabeling Procedure The basic photolabeling procedure involves initial incubation of the radiolabeled photoprobe with the protein or a crude mixture of proteins (i.e. cell lysate or tissue homogenate) for a brief period of time (15 sec to 2 min.). All experiments are usually performed on ice at 4°C to minimize hydrolysis of the gamma phosphate or phosphoryl transfer reactions. Brief (1-2 min.) photolysis with a low intensity (2000-6000 µW), hand held 254 nm UV light produces a covalent linkage within the active site. This unique photochemical property allows quick photolabeling at low intensity without damage to the protein. Immediately after photolysis, the photolabeling reaction is quenched with a thiol reagent such as dithiothreitol or beta mercaptoethanol (10 mM or greater). This scavenges all potential long lived intermediates and ensures no "pseudo" photoaffinity labeling. The photolabeled protein is then separated from the unbound photolyzed probe by several methods such as precipitation with acid, liquid organics (methanol, ethanol, acetone), ammonium sulfate, polyethylene glycol, etc. or by gel filtration or ultrafiltration. The photolabeled proteins can then be analyzed by virtually any biochemical method: SDS-PAGE, two dimensional IEF x SDS-PAGE, FPLC, HPLC etc. Quantification of the degree of photoincorporation of radioactivity can then be simply determined by any of a number of commonly used techniques such as autoradiography, imaging analysis or scintillation counting. Equipment Needed Photolabeling ice tray is a micro sample tube rack made by Belart Scienceware (http://www.bel-art.com). The rack is two piece with a lower tray which holds ice and the removal upper section which holds the sample tubes. This tray holds the hand-held lamps shown in the picture securely in place over the sample tubes with a minimum UV light path length. UV lamp is a Mineralight Model UVG-11, shortwave 254nm lamp from UV Products (http://www.uvp.com), intensity 4000µW. There is no need to remove the 254nm filter to activate aryl azides with maximum UV absorbance's in the 270-280nm range. There is more than sufficient UV light intensity in this range even with the filter in place to effectively photoactivate aryl azides upon brief irradiation (1-2 min). Make sure the sample is positioned in the tube holder so that the center of the lamp bulb is directly over the top of the opened sample tube in the tube rack. We recommend marking this tube holder spot on the ice tray rack to ensure that all samples are placed in the same place each time during photolysis. Warming the lamp for 5 minutes also helps to ensure maximum intensity. Microcentrifuge tubes 0.5-2.0 ml. Make sure the caps are opened and folded back so as not to shield the sample from UV light during irradiation. We also recommend photolyzing one sample at a time to ensure maximum reproducibility. Timing and accurate pipetting are everything in a photolabeling experiment. A few seconds or a few microliters here or there can make a difference. Make sure to keep track of time of incubation of the photoprobe with the sample as well as the time of photolysis.
Important Handling Procedures Nucleotide photoaffinity probes are supplied in absolute methanol and should be stored at -20oC. When ready to use, aliquot the required amount of probe. Remove methanol by evaporation in a speedvac vacuum concentrator or under a gentle stream of nitrogen or air in a fume hood. The dried nucleotide can then be resuspended in the appropriate buffer (minus reducing agent!!). Normal room lighting is adequate for the working environment and will not activate the probe. We make all of these compounds with the lights on. However, the photoprobe should be protected from inadvertent exposure to UV light or sunlight. When working with 32P labeled photoprobes, acrylic shielding is recommended (minimum 3/8" thick) as required to reduce exposure to radiation. Additionally, a G-M type radiation monitor should be available to insure a contamination free work area. Technical Information or General Inquiries Contact Dr.
Anjan Bhattacharyya, Ph.D.
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