Dialysis - (Jul/31/2007 )
I"m trying to find a protocol for diaylsis
is it usually about 2L of buffer for 5 mL of sample? also why do we need EDTA in the dialysis buffer? can I do it without it?
Thanks very much!
i do my dialysis without EDTA. but it may depend of the protein you're currently using.
2L for 5ml seem to met truly ok.
About EDTA in dialysis buffer - it depends on your purpose! For example addition of EDTA is necessary to prevent air oxidation of FREE SULPHYDRYL GROUPS in proteins ( otherwise possible protein aggregation will occur during long-term dialysis) which catalysed by trace amounts of metals in dialysis buffer.
About Dialysis usually dialyse 2 hour at RT then change fresh buffer and night dialysis ar 4 C. To accelerate process make desalting prosedure on Sephadex G 25 column - especially for little volumes of protein sample like 5 ml. Only 10-30 minutes
is it usually about 2L of buffer for 5 mL of sample? also why do we need EDTA in the dialysis buffer? can I do it without it?
Thanks very much!
I regularly dialyse FBS/FCS to remove Arginine, as I initiate my assay with exogenous L-Arginine. The FCS/FBS supports cell growth and there is NO detectable argininge present after dialysis. My method is below:
Use the appropriate MW cut off dialysis tubing i.e 3, 5, 10,000K ( I use 10,000K).
Some come prepared in solution, some dry. I use dry tubing which need to be hydrated overnight at +4oC.
Cut an appropriate length for your dialysis and tie one end off.
Fill tubing with solution ready for dialysis
Tie other end off (check for leaks) and immerse in solution you want to dialyse against i.e. PBS.
Leave overnight at +4oC stirring slowing on a stirrer platform.
Next day make up new PBS and immerse tubing in that at +4oC overnight
Next day do the same as above.
So dialysis is over a 3 day period. I dialyse 100ml of FCS/FBS against 3 lots of 10 Litres of PBS.
Hope this is useful
Rhombus
is it usually about 2L of buffer for 5 mL of sample? also why do we need EDTA in the dialysis buffer? can I do it without it?
Thanks very much!
Sorry forgot to say that I DO NOT ADD EDTA TO MY PBS. However dialysis is successful without it.
does anyone know the molecular weight of sarkosyl? our lab has a diaylsis tube of 10 000 cut off, don't know if that's too small?
also, i use sarkosyl in my lysis buffer, is it really necessary to use dialysis to remove it? i mean, once my protein binds to the column, and if I just wash with PBS buffer (without sarkosyl) wouldn't it all wash away anyway? because I want to use MONO Q afterwards so I can't have any anionic detergent
thanks a lot
If under sarcosyl you mean N-lauroylsarcosine sodium salt so MW- 293.4, if N-lauroylsarcosine neat so Mw - 271,4. Sarcosyl is dialyzable detergent so no problem with removal by dialysis
Here http://wolfson.huji.ac.il/purification/PDF/detergents/CALBIOCHEM_DetergentsGuideRemoval.pdf on 10-th page of brochure you will see more info about choice of detergent removal strategy. And here http://www.sigmaaldrich.com/img/assets/15402/Detergent_Selection_Table.pdf a link for properties of commonly used biological detergents. Last link provide you the main parameter critical to you is CMC ( The critical micelle concentration). If concentration of detergent is lower than CMC so micelles destroyed into low molecular species and easy removal by dialysis you reach.
thanks so much!
Here http://wolfson.huji.ac.il/purification/PDF/detergents/CALBIOCHEM_DetergentsGuideRemoval.pdf on 10-th page of brochure you will see more info about choice of detergent removal strategy. And here http://www.sigmaaldrich.com/img/assets/15402/Detergent_Selection_Table.pdf a link for properties of commonly used biological detergents. Last link provide you the main parameter critical to you is CMC ( The critical micelle concentration). If concentration of detergent is lower than CMC so micelles destroyed into low molecular species and easy removal by dialysis you reach.
a little primer on dialysis:
dialysis is a method for diluting small molecules without changing volume. you select a membrane with a molecular weight cutoff that is small enough to hold your molecule of interest in while large enough that it allows other molecules to exit into the surrounding medium. it will also allow small molecules to enter the "bag". this all works by diffusion until equilibrium is achieved. equilibrium is reached in about two hours (depending on diameter of tubing) with gentle stirring.
you can effectively dialyze in relatively low volumes. for example, say you have 100 ml of protein solution in a dialysis bag and 1000 ml of dialysis buffer. if you drop the bag into the whole solution then you will dilute the salts 10 (actually 11) times. if you were to split the dialysis buffer in half (500 ml each) and drop the bag in one for 2 hours and the other for another 2 hours you will dilute 5*5=25 times with the same 1000 ml. split the dialysis buffer into 4 equal parts (250 ml each) and you will dilute 2.5*2.5*2.5*2.5=39 times (in about 8 hours). this would leave your nacl concentration (from 150 mM nacl in pbs) at less than 4 mM and would be fine for use on the mono Q column. with lower volumes of protein solution you have even greater dilution factors.
dialysis is a method for diluting small molecules without changing volume. you select a membrane with a molecular weight cutoff that is small enough to hold your molecule of interest in while large enough that it allows other molecules to exit into the surrounding medium. it will also allow small molecules to enter the "bag". this all works by diffusion until equilibrium is achieved. equilibrium is reached in about two hours (depending on diameter of tubing) with gentle stirring.
you can effectively dialyze in relatively low volumes. for example, say you have 100 ml of protein solution in a dialysis bag and 1000 ml of dialysis buffer. if you drop the bag into the whole solution then you will dilute the salts 10 (actually 11) times. if you were to split the dialysis buffer in half (500 ml each) and drop the bag in one for 2 hours and the other for another 2 hours you will dilute 5*5=25 times with the same 1000 ml. split the dialysis buffer into 4 equal parts (250 ml each) and you will dilute 2.5*2.5*2.5*2.5=39 times (in about 8 hours). this would leave your nacl concentration (from 150 mM nacl in pbs) at less than 4 mM and would be fine for use on the mono Q column. with lower volumes of protein solution you have even greater dilution factors.
Good points!
you should not dialysed 5ml in 2 L buffer, it is not effective compared to performed in 100 ml, and change the buffer 5 times every few hours.