The literature I'm reading stated that they dialyzed their protein solution in a borate buffer (100mM borate, 150 mM NaCl, pH 9). They didn't state the volumes used, so I read up from other sources and they said that it is advisable to dialyze 1 mL of protein sample to about 200 mL of buffer. My question is, for example: If i am planning to dialyze 25 mL of sample, so that means I need 12.5L (which seems like an awful lot...is this normal?), does that mean that I need to prepare 12.5L of 100 mM borate buffer, meaning actually using 1.25mol of sodium tetraborate decahydrate (MW = 381) or roughly 476.25g of it? It just sounds like so much. I'm not sure how to go about it and I don't want to waste so much chemicals. I'd appreciate the help.
No, think about this.
You could dialyse against an equal volume to your sample. The equilibrium concentration is 50% of the starting material. Then, change the buffer - now 25%, once more 12.5% etc. This is an absurd example, but as an alternative, consider three successive change of 100X sample volume - this is equivalent to a single dialysis of 1mL to 1000 litres! More realistically, one change of 100X gives you the equivalent of 1mL to 10 litres. Same point has been made above. Buffer changes are your friend!
Lastly, you need to know why you are dialysing.
And, if you are struggling with simple buffers, look here:
www.liv.ac.uk/buffers
It can also be dialyzed in 1 litre buffer with three changes(approximate 4l buffer will be used)
The volume must be enormous if compared to that of the sample. This, is the main rule to follow. At least three changes are recommended for successful dialysis.
I recently dialyzed my 10 ml samples in 1L buffer changed 4 times.
Most of the above comments are right. 25mL sample can be divided into say 3 lots of 5- 10 ml and dialysed together in 2L buffer with 4 changes over 24 hrs.
I think an acceptable amount for dialysis buffer is to use around 300X your sample volume. Thermo sells 30mL dialysis cassettes that would be perfect for your sample size, or you could split them into two 15mL cassettes. I typically dialyze 10-12 mL of protein in a 15 mL cassette and use about 3L of dialysis buffer in total (1L, twice for 2 hrs each, then a final 1L overnight). Also, do the dialysis at 4C.
No, think about this.
You could dialyse against an equal volume to your sample. The equilibrium concentration is 50% of the starting material. Then, change the buffer - now 25%, once more 12.5% etc. This is an absurd example, but as an alternative, consider three successive change of 100X sample volume - this is equivalent to a single dialysis of 1mL to 1000 litres! More realistically, one change of 100X gives you the equivalent of 1mL to 10 litres. Same point has been made above. Buffer changes are your friend!
Lastly, you need to know why you are dialysing.
And, if you are struggling with simple buffers, look here:
www.liv.ac.uk/buffers
My general rule is that buffer volume has to be 10X-20X the sample. Dialysis duration (O/N rather than few hours), temperature (I assume yuo operate at about 4°C) and buffer changes affect the buffer exchange and are to be taken into consideration. In general, the more the better. You should also consider the buffer your sample is in and if you are simply diluting the original buffer or completely exchanging buffers, the latter case needing more time and buffer changes to speed up the process.
The volume of buffer should be about 100 times that of the sample to be dialyzed, if the volume of your sample is very large, it is necessary to concentrate to reduce the volume of buffer used. A good reference is: Protein Purification Protocols by Paul Cutler, Chapter 11: Making and Changing Buffers.
The volume of buffer for dialysis is determined by two factors; how high dilution you should like to reach and how many steps you would like to employ. In general, if dilution higher than 100-fold is the aim, you should plan more than one dilution step. In your example the required dilution is 200-fold. In this case you a small volume (1 ml) can be dialysed in 200 ml buffer, however, the 25 ml sample is too much for such a one-step dialysis. With 25 ml sample it is recommended to divide the sample into smaller portions (it will speed up the dialysis time too), and employ two consecutive dialysis steps; for instance 5 x 5 ml dialysis in 5 x 100 ml buffer (first dialysis step) and repeat it once. In this case your two-step dilution protocol will result in 20-fold times 20-fold (=400-fold) dilution - and you use only 1000 ml of buffer.
If I divide the 25 ml into different dialysis tubings, would it be okay to place all these tubings into 1 whole container with 2.5L? Would the calculations still be the same such that the new concentration will simply be divided by 100? Or do I need to setup separate containers for each dialysis tubing?
Dear Christine, "speed" of dialysis is highly depending on the surface-to-volume ratio of the sample. If you divided your 25 ml sample into 5 x 5 ml sample, you will increase the surface of the very same 25 ml sample volume; thus the surface-to-volume ratio will increase and the "speed" of dialysis (the speed of reaching the equilibrium) will be higher. However, you will reach the very same final dilution (in your example the 100-fold dilution) in all the three following cases: (1) 25 ml sample in 2500 ml dilution buffer; (2) 5 x 5 ml sample in 2500 ml dilution buffer, (3) 5 x 5 ml sample in 5 x 500 ml dilution buffer. Never forget to apply a mild (!) stirring in the dialysis buffer.
One does not need to divide the sample to increase the surface. Simply use a smaller dialyzing bag (lower diameter). Particularly convenient are thicker wall (e.g. 1 cm) old fashion “Visking” and not the new fancy ones with different MW cuts. The later do not work! Thick walls prevent osmosis and resulting in lower increase of the volume inside. Also important is the volume ratio between inside and outside. This does not need to be large when somebody changes the dialyzing solution every few hours. On this way dialysis time can be much shorter. I suggest the volume ratio of about 1 to 5(-10) –folds with the three-four changes every 3-4 hours and the last convenient one overnight. This should be more than sufficient to be sure that dialysis is O.K.
I have usually dialyzed a 100 mL sample in 1L buffer with about three changes, that is a total of 3L buffer on a magnetic stirrer so that the equilibrium on the membrane surface does not form, and dialysis goes on. If the purpose is simple desalting, one could use a column like Sephadex G-25, or centrifugal conentrators like Centricon. If the purpose is buffer exchange, then dont forget to dialyze against distilled water first before dialyzing against the required buffer to prevent precipitation of the protein. If carried out in 2L beaker/1L measuring cylinder with stirring, you dont need to aliquot your sample.
For Vivek, Regarding Buffer exchange: Dialyzing against water will precipitate out many proteins..which protein did you try this with?
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