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HDL dialysis | Magnus | 2001-10-22 | Click here to register. |
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I want to remove 50 mM tris and 2 mM EDTA from a batch of high density lipoprotein (HDL) also containing 154 mM NaCl. I am planning to use PBS as the exchange buffer. HDL particles are between 6-10 nm in size. What MWCO should I use? This shouldn't affect the volume of my sample, would it? I'm planning to use Spectrum's Dispodialyzers. How long does it take for a 1 ml dialyzer tube to equilibrate in a 1 l exchange volume? Does this depend on MWCO or the membrane type? What to consider when chosing membrane types? Do certain molecular species stick to the membrane, in my case I worry of course for the phospholipids in my lipoprotein. How exact are the pores? Is it wise to chose an MWCO with a significant margin to the protein you want to stay on the inside?
Thanks in advance
Magnus Johnson, MD
Gothenburg, Sweden | | |
| dave | 2001-10-22 | Click here to register. |
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 | Since you are planning on using a dispodialyzer, a nice 15K RC membrane would probably be a good choice (the 6nm diameter is about 50K MWCO).
If the osmotic pressures of your sample and the outside buffer are the same, the sample volume will remain relatively constant. If the osmotic pressures are different then the volume will probably change some. (You can estimate the osmotic pressure by taking the sum of the concentrations of each ion in solution and adding the sum of the concentrations of the non-ionic species.)
The exchange volume doesn't significantly affect the equilibration time, just the final concentrations. For small ionic species, generally overnight or 6 to 12 hours is used. It depends upon how close equilibrium you need to be.
The time for dialysis depends upon the composition of the membrane, the MWCO of the membrane, the sizes of the ions and other species that are to pass through the membrane and how close to equilibrium you need to be. Dialysis experiments are usually run extra long to make sure that they are near equilibrium.
Yes, some molecule stick to the membrane. These tend to be those which are only marginally soluble or that have non-polar regions (like lipids). The best way to evaluate the sticking is to try a readily available compound that you expect to be similar to your sample compound.
To minimize the sticking you can try to saturate the membrane. When people worry about proteins sticking, the common remedy is to rinse the membrane with a solution of bovine serum albumin (which sticks very well) and then with buffer (to remove any free BSA) and then use the membrane. I assume that either BSA or a lipid of some sort could be used if you anticipate stickiness troubles.
It is usually wise to pick a pore size that is 2 to 3 times smaller than the molecule to be retained. (It is also recommended that the pore size be 2 to 3 times larger than any molecule to be removed. This is why dialysis is not used for the determination of molecular weights.) |
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| Magnus | 2001-10-23 | Click here to register. |
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| Thanks for the quick and comprehensive reply, Dave. One follow-up if I may: if I decide to perform two serial dialyses, should I use the same dispodialyzer tube for the second round or should I use a new tube?
Thanks again,
Magnus |
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| dave | 2001-10-23 | Click here to register. |
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| If you mean put the sample in the tubing and then dialyze against one buffer and then another, there's no reason to use a second dialyzer. Just move the dialyzer from one buffer tank (a.k.a. beaker) to another. This saves a step handling the sample and minimizes any losses from absorption to the membrane.
The only time when you might want to change dialyzers is when the outside solution (the replacement buffer) has a lot of muck in it that adsorbs to the membrane. I haven't heard of anyone dialyzing against a solution with a lot of adsorbing proteins but there's nothing to prevent it either.
If you are going to manipulate the sample some other way between the two dialysis steps, then you'll need to decide whether or not to use a new membrane. It will depend upon when the trace of sample left in the dialysis sack will be bad when mixed with the manipulated sample. |
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