Slide-A-Lyzer

Slide-A-Lyzer Slidalyzer or slide a lyzer used for dialysis, sold from thermo scientific

http://www.piercenet.com/browse.cfm?fldID=E1D032C4-B084-2D48-7ECC-27D9E94B41F4 Instructions http://www.piercenet.com/instructions/2162132.pdf

Typical dialysis buffer: PBS or TBS Large flask
 * large flask not contaminated with soap for use with dialysis last seen in cabinet below Bart's bench

A. Hydrate Membrane 1. Remove the cassette from its protective pouch. To prevent membrane contamination, handle the cassette by the plastic frame only. Do not touch the membrane with ungloved hands. The cassette may be placed upright on the bottom end on a flat surface. 2. Immerse cassette in dialysis buffer for 2 minutes to hydrate membrane (Bart and I have often just used H20 instead of dialysis buffer). It may be necessary to hold the cassette under the surface for the hydration step as the air inside the cassette may cause it to float sideways. Note: Hydration increases membrane flexibility and allows it to adjust more readily to the sample loading and removal of excess air. 3. Remove cassette from buffer. To remove excess buffer, gently tap the cassette on a paper towel. Turn the cassette upside down and tap again. Do not blot the membrane. B. Add Sample Note: The minimum sample volume for the 3, 15, 30 and 70mL cassette is approximately > ⅓ of the cassette’s maximum volume or leakage can occur. For the 0.5mL cassette, the minimum sample volume is ≥ 250μL. Caution: To avoid injury from the needle, do not remove the needle’s plastic sheath until ready to use. The cassette is designed for 18 gauge, 1-inch beveled needles (21-gauge, 1-inch beveled needles also may be used). 1. Fill the syringe with the sample, leaving a small amount of air in the syringe. 2. Penetrate the gasket through a syringe port at the cassette’s corner. Insert the needle so that the sharp open end is facing towards the middle rather than the sides to avoid tearing the membrane. Overextending the needle into the sample chamber may puncture the membrane (Figure 2). Slowly extend the needle minimally into the cavity so that the open end of the needle is barely visible (Figure 3). 3. Inject approximately half of the sample. For samples with high protein concentrations (e.g., 10 mg/mL), avoid foaming by filling the cassette slowly. 4. Withdraw some air from the cassette by pulling back on the syringe piston and then inject remaining sample. 5. With the needle inserted into the cassette cavity, withdraw remaining air to compress the membrane windows so the sample contacts the greatest possible membrane surface area. Use caution to prevent the needle from contacting the membrane. A small amount of air left inside the cassette will not significantly affect dialysis efficiency. 6. Remove needle from the cassette while retaining air in the syringe. The gasket will reseal and the membrane cavity will contain minimal or no air. Place a mark on the cassette corner with a permanent marker to note which needle port was used. C. Dialyze Sample 1. Float cassette vertically in the dialysis buffer and stir gently to avoid creating a vortex that might pull the cassette down in contact with the stir bar. 2. Dialyze for an amount of time sufficient to remove low molecular weight compounds for the specific downstream application. A typical dialysis procedure is as follows: dialyze for 2 hours at room temperature or 4ºC; change the dialysis buffer and dialyze for another 2 hours; change the dialysis buffer and dialyze overnight. Use the dialysis buffer at a total of at least 300 times the sample volume throughout the course of the dialysis procedure. -May want to concentrate sample by adding BSA to surface of membrane. May want to add more BSA to this to concentrate the volume further. This can be repeated (possibly with fresh BSA as well) until you have the volume desired). D. Recover Sample Note: Avoid penetrating guide ports more than once to prevent gasket coring and subsequent sample loss. 1. Penetrate gasket with the needle through an unused syringe guide port and slowly inject air into the sample chamber to maximum allowed sample volume to separate membranes, which prevents membrane needle puncture. (Figure 4). 2. With the needle in place, turn the unit so that needle is at the bottom. Allow sample to collect near the port and withdraw sample into the syringe (Figure 5).