We normally photolabel purified proteins and tissue homogenates with a final photoprobe test concentration of 10-15 然 in a 40 痞 final test volume. This allows plenty of working volume for the addition of activators and inhibitors of nucleotide binding (i.e. metal ions, cofactors, chelators, competing nucleotides, etc.). Usually the purified protein (0.5-1 痢 per test) or tissue homogenate (20-50 痢 per test) is incubated for some period of time (5-30 min) on ice with the activator or inhibitor in a 30 痞 reaction volume. Afterwards, a 10 痞 aliquot of the stock photoprobe solution is added to this reaction, vortexed gently or mixed with pipeting, allowed to incubate for 30 sec and irradiated at 254 nm with a hand-held UV lamp for 60 sec. With this in mind, we can now calculate how much photoprobe we are going to need to do this experiment. 1) Determine the volume of stock photoprobe solution needed. Using these conditions let's assume that we wish to determine the effects of increasing Mg2+ concentration on [g32P]8N3ATP photolabeling of purified pyruvate kinase (PK). We are going to test Mg2+ at 5 different final test concentrations - 50, 100, 250, 500 and 1000 然. Therefore, we will need enough photoprobe for 5 tests plus one control with no Mg2+. Normally we bump this one additional test just in case we make a mistake or have an accident. This gives us a extra aliquot of photoprobe to play with. That makes 7 test total at 10 痞 of photoprobe solution per test for a total probe volume of 70 痞 (7 test x 10 痞/test = 70 痞). 2) Determine the concentration of the stock photoprobe solution. If we desire a final [g32P]8N3ATP test concentration of 10 然, we can simply multiply the desired test concentration by the total test volume divided by the volume of the photoprobe aliquot added (the dilution factor) to determine the actual concentration of the stock photoprobe solution needed (10 然 x 40/10 = 40 然). Therefore, we will need a total of 70 痞 of a stock 40 然 [g32P]8N3ATP solution to perform this experiment. 3) Determine the nanomoles of photoprobe needed. Knowing the desired photoprobe test concentration and the volume of stock photoprobe solution needed, we can now calculate the nanomoles of [g32P]8N3ATP we are going to actually need to complete this experiment. This is determined by multiplying the stock [g32P]8N3ATP concentration by the total volume of photoprobe required (40 nmol/ml x 0.07 ml = 2.8 nmol) Therefore, to perform this experiment we are going to need a total of 2.8 nmoles of [g32P]8N3ATP. 4) Determine the volume of concentrated stock 32P]8N3ATP to dry down for the experiment. Lets assume this months [g32P]8N3ATP (in absolute methanol, stored at -20oC) is 563 然 (specific activity of 21.5 mCi/痠ol on 2/15/99). To calculate the volume of this concentrated stock 563 然 [g32P]8N3ATP we are going to need, simply divide the nmoles of photoprobe required by the concentration of the stock photoprobe (2.8 nmol divided by 563 nmol/ml = 0.00497 ml). Therefore, we need to dry down 5.0 痞 of the concentrated 563 然 stock [g32P]8N3ATP to in order to complete this experiment. This is best done in a vacuum concentrator or under a gentle N2 or air stream. After drying down the 5 痞 of stock 563 然 [g32P]8N3ATP to remove the methanol, we are now ready to resuspend our photoprobe in our working buffer solution and begin photolabeling. Money Saving Tips If you wish to conserve on radiolabeled photoprobe, we suggest you dilute the radiolabeled nucleotide with nonradiolabeled compound. For example in the experiment described above if we want to decrease the amount of [g32P]8N3ATP required for the experiment but maintain the same 10 然 final photoprobe test concentration, we could use 1.4 nmole of [g32P]8N3ATP and 1.4 nmole of cold 8N3ATP (product # AT02). Of course this is going to dilute the specific activity of the photoprobe solution in half but normally this is not a major problem given the fairly high specific activity of starting material. 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. You may have to wait an extra day or two for the autoradiogram to develop but this can be reduced by sandwiching the gel and film between 2 enhancing screens instead of just one. Precautions Always prepare your photoprobe solutions immediately before use. Never store photoprobe solutions in aqueous buffer solutions for extended periods of time (greater than 2-3 hours) even if frozen. Azido based photoaffinity compounds are extremely stable when stored in absolute methanol at -20oC but not when they are stored for extended periods of time in aqueous buffers. Therefore, it is always best to make photoprobe solutions just before you are ready to perform the experiment. While actually performing the experiment, we recommend you keep the photoprobe solution on ice in a beta block or some sort of shielded holder. To Calculate Photoprobe Specific Activity on a Given Day Knowing the specific activity of the radiolabeled photoprobe on a certain day (given on the technical data sheet supplied with each order) the following formula can be used to calculate the specific activity on the day in question. Lets assume that the experiment described above was performed on 2/21/99. The specific activity of the 563 然 [g32P]8N3ATP was 21.5 mCi/痠ol on 2/15/99). To calculate actual specific activity (SA) on the date of the experiment we can use the following known values and constants for 32P to calculate the specific activity. Reference date of photoprobe manufacture (t=0): 2/15/99 Date of actual experiment: 2/21/99 Number of expired days (t): 6 days Radioactivity decay constant (l) for 32P: -0.0485 Initial Specific Activity (SAO): 21.5 mCi/痠ol Formula: SA0 = SAt=xe-lt Calculation: 21.5 mCi/痠ol = SAt=6e(-0.0485)(6) SAt=6 = (21.5 mCi/痠ol)(0.7475) SAt=6 = 16.1 mCi/痠ol on 2/21/99
For additional information on how to perform basic radioactivity calculations for biochemical and pharmacological experiments see the Radioactivity Calculator at http://www.graphpad.com/calculators/radcalc.cfm Technical Information or General Inquiries Contact Dr.
Anjan Bhattacharyya, Ph.D.
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