Keating:Experimental Protocols:Capillary Electrophoresis

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Instructions for using Beckman PACE MDQ capillary electrophoresis

4/21/05

1.Prepare buffers and samples choose a buffer or set of buffers to use as the running and sample buffers ideally ~2 pH units above pI of protein (if known) when in doubt, start with borate buffer pH 8.0 concentration 20-200mM (start with 50mM) salt in buffer will interfere with conductivity and separation 3 vials of each buffer (~1.5ml each) – top with orange caps don't fill past the shoulder of the vial

samples should be approximately 50uM in a minimum volume of 50ul of sample buffer (same as running buffer) not much of the sample will be injected and the majority of the sample can be recovered at the end of the experiment place samples in PCR tubes in plastic vials with springs, topped with gray caps also prepare water, 0.1M HCl, 0.1M NaOH, methanol, and waste vials filter all samples and buffers with 0.22um filters make fresh buffer aliquots for every ~10-20 runs / 2-3 weeks

2.Open PACE MDQ software program software is very similar to the HPLC software instrument should already be on

3.Turn on UV lamp to warm up for at least 15 min 4.Make a directory for yourself in D:\Users\CE 5.Sign in the log book

6.Change cartridge and/or capillary if desired. in software, manual control – press load to move vial trays forward open tray and cartridge cover unscrew the insertion bar keeping the cartridge in place remove the cartridge follow instructions in the installation and maintenance manual to change the capillary reverse instructions to replace

6.Place sample and buffer vials in buffer and sample trays pay attention to where the tray positions for writing the methods below is the standard positions for the minimum buffers needed

samples placed in the back sample trays can be kept at particular temperature, but can also be placed in buffer trays (only RT)

7.Methods use examples in D:\Users\CE\General Methods\ if it's the first time using the capillary ever or in several months, start with condition.met capillary to go through extensive rinsing otherwise use wash.met to do a quick rinse for separation methods, use bufferXaYb.met where Xa is position of rinse buffer vial and Yb is position of separation buffer vial separation methods include a few washes, injection of the sample, and separatation of the sample under voltage method files assume injection of buffer as the sample (baseline) after all runs have been performed, use shutdown.met to give the capillary a final rinse, includes turning off the lamp any of methods can be modified change absorbance wavelenth, sample/buffer positions, length of time for separation or washes, etc please save in your directory's methods folder to run a single method, press the blue arrow save the data file in your directory's data folder

8.Sequences to run multiple samples, use a sequence file to specify the method, data file, and sample position of each sample for examples, look in D:\Users\CE\Nora\Sequences you need to name the data files by typing in the sample, including the entire path with your directory press the green arrow to start the sequence sequence can be modified in the later steps and saved while running

Notes if window pops that coolant is low, refill coolant open bottom panel connect tube attached to syringe pour in coolant in 5ml increments, just let flow in until level of coolant in viewing tube is between the black lines