protein dialysis - i think i just cant understand it (Apr/12/2006 )

there is noone who have done it in our lab and we have this dialysis tubings from a company who doesnt answer to my mails... ...ive read some web pages but none of them states the protocol clearly....i just cant imagine the procedure....please can you tell me how you do it? thanx a lot!
sorry forgot to mention i want to remove urea from my protein.

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hi kathy ! here is the protocol to first activate your dialysis tubing.
1. cut the dialysis tubing
2. keep the tubes under running water for 4-6 hrs.
3. keep the tube in a solution containing 100mM NaHCO3, 10mM EDTA-Na salt (pH 7.0) at 60 C for 2 hrs with mild agitation ( three changes).
4. wash the tubes with triple distilled water several times.
5. you can you the tubes immediately or store it at 4 C in TDW containing 0.02 % sodium azide as a preservative.
6. if you want to use it immediately then close one end of your tube with a clip transfer your solution to be dialysed into the tubing and close the other end too forming a bubble like structure with some space for the buffer against which you are dialysing can enter (remember your solution is concentrated and buffer enters the tube holding your solution.)
7. dialyse against your buffer of interest/ or TDW (chilled).
8. keep the entire set up at 4 C in a refrigirator.
i hope this will do
all the best

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Could you give some more details, which sort of tubes (company)? We´re working with slid-a-lyzer mini from pierce! Maybe I could help you

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we have three methods for preparing dialysis tubing. depending on the protein's sensitivity to potential contaminants (which will ultimately dialyze away).

the dry tubing is impregnated with something to keep it from getting brittle (glycerol, i think) and may also be carrying some cations with it. so...

if we need to make sure that there are no contaminants present:

cut tubing to length greater than necessary for volume of solution (volume/length is given on box) (we often prepare a batch of tubing and store it);

place in beaker with deionized water and edta (we often just throw in some solid edta) and bring to boil;

pour off edta/di water and rinse with fresh di water, add di water and bring to boil, repeat;

final rinse with di water.

tubing should be open and ready to use. if you prepare a batch then you can cut off the length that you need and store the rest either in di water (short term) or 10 % etoh (long term) at 4C.

if you don't need to be that stringent then you can skip the edta step.

if the possible contaminants don't matter or for a quick preparation you can just hydrate and open the tubing with running di water.

we normally don't use clips, we tie knots in the ends of the tubing. use 2 knots on each end and pull them tight from the cut tubing side of the knot (not the filled or to be filled side, that will stress the cellulose). after introducing your sample into the tubing, leave a air pocket then tie off the end. this pocket will allow the tubing to float near the top of the dialysis solution and will keep it away from the stirring bar.

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We also use Pierce Slide-a-Lyzers. They're great -- I much prefer them to tubing.

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Homebrew-me too. dialysis with tubing is a PIA

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slide-a-lyzers are nice but they are only good for up to 30 ml and are relatively expensive (for a poor, underfunded lab). we routinely dialyze volumes up to 250 ml (and sometimes more). for that we need to use tubing or diafiltration.

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thank you very much for your replies!

Ms olli, the company is wako...maybe you never heard of it i guess its japanese...since im in japan, but maybe not i dont know....all their catalogues are in japanese so... ..anyway i am also writting to them since they have two packages here in the lab one size 8 and other size 20.. ..since their webpage doesnt explain anything i cant understand the difference....maybe MWt cut off but im not sure...any idea?? they are called "dialysis membrane"
we also have here seamless cellulose tubing....from other japanese company....



i need to dialyze around 1 ml sample..... ....is it possible using one of those tubing kinds??

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hi kathy ! you get microdialysis tubes from amersham. you can even prepare your own microdialysis setup with the tubing you have.it is very simple.
1. since your sample size is 1ml. you can use 2ml. centrifuge tubes to hold your sample. cut the lid of the centrifuge tube so that you can wrap/ fasten the activated tubing onto its mouth tightly with the help of a thread.
2. activate the tubing as i have mentioned earlier. wear gloves while handling.
3. cut the tubing in the form of a circle (only one of its side needed) so that it can cover the entire mouth of the centrifuge tube and you can tie it with the help of a clean thread with sample inside the tube. take care so that the sample does not leak.
4. fix the tube into a float and dialyse the sample against the buffer of your choice with constant stirring on a magnetic stirrer.
5. place the entire set up in a refrigirator if your protein is heat unstable.
the entire set up is illustrated in the form of a diagram in this attachment
all the best

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Spectrum makes a small diameter dialysis tubing (used to be called 1/4 inch, now it's called 4 or 5 or 6mm, i forget which) that we use for smaller volumes like 1 ml and even less. but shiva keshava's method looks useful and slide-a-lyzers and similar products are good for your volume.

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