Dialysis is an essential method in protein biochemistry when purifying peptides, native or recombinant expressed proteins. Dialysis can also be used to concentrate or desalt protein solutions, remove low molecular weight products or simply to exchange the buffer system.
The Slide-A-Lyzer dialysis cassettes contain a low-binding cellulose membrane and are an easy to use, leak-free dialysis system. There is no need to boil dialysis membranes in EDTA solutions, tie knots or close membranes ends with dialysis clips, which might leak or open during dialysis. Before use, the cassettes are only hydrated for 2 min in buffer. Due to the high surface area/sample volume ratio, buffer exchange is fast. The Slide-A-Lyzer dialysis cassette are available in packs of 6, 8, or 10 individually wrapped single frames, or as kit which includes the cassettes, an equivalent number of syringes, needles and float buoys. Cassette sizes ranges are: 0.1-0.5 ml, 0.5-3 ml, 3-12 ml and 12-30 ml, with a variety of color-coded exclusion sizes (cut-offs): 2, 3.5, 7, 10 or 20 kD. Frames with a 10 kD cut-off are also available with gamma irradiation for dialysis of sterile samples, like cells, microorganisms, viruses, DNA or RNA. Cassettes float with the attached buoys (a white one for cassettes from 0.5-3 ml, a grey one for cassettes from 3-12 ml) or with an internal buoy for the 12-30 ml cassette.
Care should be taken not to touch the membrane when handling the cassettes. Also, excess buffer after hydration should be removed by tipping the frame on a clean tissue to clearly visualize the injection of the sample. Before injecting samples, I tested the leakiness of the cassettes by injecting purified water and tapping the cassette on a dry tissue. At the same time, I could see if the needle was tightly screwed to the syringe and did not disconnect under pressure when injecting the sample. When injecting or withdrawing solutions, it is essential to always use a different port and make a note of the port number. Also the needle tip should only be moved so far into the port that it does not touch the membranes (which can cause damage). There are detailed, pictured instructions in the manual which show how to hold the cassettes during sample injection and withdrawal. When filling the syringe with small sample volumes, I always draw some air into the syringe; this air stays as a bubble on top of the sample. Injecting the total volume of the sample is then possible, leaving the needle dead volume filled with air. To ensure spreading of the sample over the entire membrane surface, the air in the chamber should be withdrawn. Make sure you hold the pistol in the same position after withdrawal of the air from the chamber and while moving the needle from the port in order to avoid re-injection of the air. Before dialysis, I attach the buoy to the cassette so that the ports I injected are on the top. I then test for leakage from the ports by tapping the cassette on a fresh, dry tissue before dialyzing.
I have used these dialysis cassettes to neutralize the buffer of protein-A purified rabbit IgG antibody (about 110 kD). The antibody was eluted with citrate buffer pH 3.0-2.5 and than dialyzed against PBS in frames with 10kD cut-off. I generally dialyze for 4 h with 2 buffer changes, in addition to an overnight dialysis in the cold room (3-12 ml cassettes in a 2l beaker of buffer). I have never experienced any major leaks during the dialysis using the cassettes. In my experience, a film of protein always sticks on the membrane after dialysis and withdrawal of the sample; due to the higher surface area of these cassettes, protein loss might be higher, especially when highly concentrated samples or smaller volumes are dialyzed.
The Slide-A-Lyzer dialysis cassettes are an easy to use and efficient dialysis system for a variety of dialysis applications. The manual provides all necessary information for using the membranes, but care should be taken in choosing the right cut-off and frame size (i.e. in order to avoid loosing low molecular weight proteins of interest or minimize the protein loss). It is better to choose a lower cut-off size when exchanging buffer systems (e.g. to neutralize pH or remove salt); for a protein solution, a 2 or 3.5 kD cut-off will avoid protein loss. When the dialysis is used to eliminate lower molecular weight molecules (e.g. peptides), a cut-off just below the desired MW should be used.