(Optional) Concentration of the SampleDecide which, if any, samples will require concentration. For example, the crude extract and ammonium sulfate pellet fraction may not need concentration but the ion exchange pooled fractions may. Since you have already determined the protein concentration of your fractions, you can do some calculations to determine if any of them needs concentrating. For SDS PAGE analysis, the sample volume in each well should contain approximately 20-100 µg of protein, if there are many proteins present, but should only contain 1-20 µg if only 1-2 bands is expected. Do not concentrate the entire sample, only what you will need for electrophoresis and HPLC, etc. There are many ways to concentrate proteins. Most involve the removal of water and salts by using a membrane with a defined pore size, similar to a dialysis membrane, through which the low molecular components are removed, leaving the more concentrated protein solution behind. Water and salts can be forced out of the protein solution by physical means such as centrifugation or pressure and also by osmotic forces, for example, by placing the protein solution in a dialysis bag and covering the bag with a substance such as polyethylene glycol that has a higher affinity for water than does the protein. We will be using small ultrafiltration devices from the Millipore Corporation, the Ultrafree-MC, with a molecular weight exclusion limit of 10,000 Daltons. Refer to product literature on these devices for specific instructions. Each ultrafiltration device is a microfuge tube containing an upper chamber with a filter membrane forming the bottom of the upper chamber. The sample is loaded into the top chamber of the tube. As the sample spins in the microfuge, the buffer is forced through the filter, while the sample protein, which is too large to pass through, remains above the filter. Note that the pore size is the same as that for dialysis in purification (Procedure 4.3).
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