Knight:Dialysis: Difference between revisions
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==Purpose== | ==Purpose== | ||
To change buffers in which your protein resides. | To change buffers in which your protein resides. | ||
<font color=red>Note that although this method should work, I find that I lose my protein using this approach. Try [[Knight:Centrifuge desalting]] instead.</font> | |||
==Materials== | ==Materials== | ||
*Slide-A-Lyzer MINI Dialysis Unit from Pierce ([http://www.piercenet.com/dialysis/mini.html video], [http://www.piercenet.com/files/0803as4.pdf manual], [http://www.piercenet.com/products/browse.cfm?fldID=04010165 product page], [http://www.piercenet.com/Objects/View.cfm?type=Page&ID=85F6FD92-10E3-495B-A256-52CAA7E93C27#Products applications guide]) | *Slide-A-Lyzer MINI Dialysis Unit from Pierce ([http://www.piercenet.com/dialysis/mini.html video], [http://www.piercenet.com/files/0803as4.pdf manual], [http://www.piercenet.com/products/browse.cfm?fldID=04010165 product page], [http://www.piercenet.com/Objects/View.cfm?type=Page&ID=85F6FD92-10E3-495B-A256-52CAA7E93C27#Products applications guide]) | ||
**''In general, the molecular weight cutoff for your column should be about half the molecular weight of your protein of interest.'' | |||
*dialysate (i.e. destination buffer) | *dialysate (i.e. destination buffer) | ||
| Line 12: | Line 13: | ||
#'''Optional:''' to remove contaminating glycerol from the dialysis unit, dialyze the unit in 1L of DI water for 15 mins. (Glycerol content: <3% in 3K, ~15% in 7K and ~23% in 10K MINI) | #'''Optional:''' to remove contaminating glycerol from the dialysis unit, dialyze the unit in 1L of DI water for 15 mins. (Glycerol content: <3% in 3K, ~15% in 7K and ~23% in 10K MINI) | ||
#'''Optional:''' to remove contaminating metals, dialyze 15 minutes against 1 L 1 mM EDTA. (Metals present in a 3K, 7K or 10K MINI; 2 ppb iron, 5 ppb magnesium, 1.5 ppb nickel, 0.2 ppb zinc, 0.2 ppb copper, 0.5 ppb chromium and 0.3 ppb cadmium) | #'''Optional:''' to remove contaminating metals, dialyze 15 minutes against 1 L 1 mM EDTA. (Metals present in a 3K, 7K or 10K MINI; 2 ppb iron, 5 ppb magnesium, 1.5 ppb nickel, 0.2 ppb zinc, 0.2 ppb copper, 0.5 ppb chromium and 0.3 ppb cadmium) | ||
#Apply sample with a standard pipette. | #Apply sample with a standard pipette. | ||
#*Sample volume should be between 10-100 μL. | #*''Sample volume should be between 10-100 μL.'' | ||
#Cap the dialysis unit and place in a floatation device. | #Cap the dialysis unit and place in a floatation device. | ||
#Add 0.5-1L of dialysate to beaker. | #Add 0.5-1L of dialysate to beaker. | ||
| Line 20: | Line 20: | ||
#Place the beaker in ice or in a cold room and on a stir plate. | #Place the beaker in ice or in a cold room and on a stir plate. | ||
#Place the float with dialysis unit in the beaker so that the bottom of the dialysis unit is in contact with the dialysate. | #Place the float with dialysis unit in the beaker so that the bottom of the dialysis unit is in contact with the dialysate. | ||
#*''The volume level of the sample should be higher than the dialysate to avoid hydrostatic pressure forcing dialysate into the unit (thereby diluting the sample).'' | |||
#Use a low speed setting on the stir plate to avoid submerging the unit. | #Use a low speed setting on the stir plate to avoid submerging the unit. | ||
#Equilibrate for 2 hours. | #Equilibrate for 2 hours. | ||
#Collect the sample from the corner of the Slide-A-Lyzer unit. | #Collect the sample from the corner of the Slide-A-Lyzer unit. | ||
==Notes== | |||
*[http://www.piercenet.com/Objects/View.cfm?type=Page&ID=1496C58D-6E13-477C-972F-72C18AB87F66#Concentration Concentrating samples via dialysis]. | |||
Latest revision as of 13:41, 5 October 2006
Purpose
To change buffers in which your protein resides.
Note that although this method should work, I find that I lose my protein using this approach. Try Knight:Centrifuge desalting instead.
Materials
- Slide-A-Lyzer MINI Dialysis Unit from Pierce (video, manual, product page, applications guide)
- In general, the molecular weight cutoff for your column should be about half the molecular weight of your protein of interest.
- dialysate (i.e. destination buffer)
Procedure
- Wear gloves to prevent contamination.
- Optional: to remove contaminating glycerol from the dialysis unit, dialyze the unit in 1L of DI water for 15 mins. (Glycerol content: <3% in 3K, ~15% in 7K and ~23% in 10K MINI)
- Optional: to remove contaminating metals, dialyze 15 minutes against 1 L 1 mM EDTA. (Metals present in a 3K, 7K or 10K MINI; 2 ppb iron, 5 ppb magnesium, 1.5 ppb nickel, 0.2 ppb zinc, 0.2 ppb copper, 0.5 ppb chromium and 0.3 ppb cadmium)
- Apply sample with a standard pipette.
- Sample volume should be between 10-100 μL.
- Cap the dialysis unit and place in a floatation device.
- Add 0.5-1L of dialysate to beaker.
- Add stir bar.
- Place the beaker in ice or in a cold room and on a stir plate.
- Place the float with dialysis unit in the beaker so that the bottom of the dialysis unit is in contact with the dialysate.
- The volume level of the sample should be higher than the dialysate to avoid hydrostatic pressure forcing dialysate into the unit (thereby diluting the sample).
- Use a low speed setting on the stir plate to avoid submerging the unit.
- Equilibrate for 2 hours.
- Collect the sample from the corner of the Slide-A-Lyzer unit.
