Daniel:Protocols/AmiconUltra: Difference between revisions
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Amicon Ultra Centrifugal filters are microcentrifuge filters that allow you to purify and buffer exchange various MW cutoff biomacromolecules. They use a MW cutoff filter, say 10 kDa, to allow dialysate through while not passing the biomacromolecules. They can be used for buffer exchange via the following general protocol: | Amicon Ultra Centrifugal filters are microcentrifuge filters that allow you to purify and buffer exchange various MW cutoff biomacromolecules. They use a MW cutoff filter, say 10 kDa, to allow dialysate through while not passing the biomacromolecules. They can be used for buffer exchange via the following general protocol: | ||
<gallery perrow=2 heights=300px widths=400px> | |||
File:CentrifugalFilters-Purification.png|Purification/Concentration | |||
File:CentrifugalFilters.png|Buffer Exchange | |||
</gallery> | |||
==Protocol-Filtration== | ==Protocol-Filtration== | ||
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<ol> | <ol> | ||
<li>Add X uL sample and new buffer to 500 uL total volume</li> | |||
<li>Spin down at 14000 x g for appropriate time (15 minutes for 22 uL volume)</li> | <li>Spin down at 14000 x g for appropriate time (15 minutes for 22 uL volume)</li> | ||
<li>Discard flow through and add 500 uL new buffer</li> | <li>Discard flow through and add 500 uL new buffer</li> | ||
<li>Repeat steps 2 and 3 as many times as desired</li></ol> | <li>Repeat steps 2 and 3 as many times as desired</li></ol> | ||
===Dialysis Table=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#FFCC66;font-size:12pt;font-weight:bold" align="center" | |||
| width="120" height="30" | Starting Sample Volume | |||
| width="110" | 500 uL dilution | |||
| width="110" | Original Buffer Concentration | |||
| width="110" | Centrifuge Time | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 50 | |||
| align="center" | 10 | |||
| align="center" | 10% | |||
| 15 min | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" | |||
| align="center" height="15" | 20 | |||
| align="center" | 25 | |||
| align="center" | 0.4% | |||
| 15 min | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 20 | |||
| align="center" | 25 | |||
| align="center" | 0.016% | |||
| align="center" | 15 min | |||
|} | |||
==Spin Times and Concentration== | |||
[[Image:AmiconSpinTimeTable.png|800px]] |
Latest revision as of 21:52, 13 May 2017
Amicon Ultra Centrifugal Filters[edit]
Amicon Ultra Centrifugal filters are microcentrifuge filters that allow you to purify and buffer exchange various MW cutoff biomacromolecules. They use a MW cutoff filter, say 10 kDa, to allow dialysate through while not passing the biomacromolecules. They can be used for buffer exchange via the following general protocol:
- CentrifugalFilters-Purification.png
Purification/Concentration
- CentrifugalFilters.png
Buffer Exchange
Protocol-Filtration[edit]
- Add X uL sample and buffer to 500 uL total volume
- Spin down at 14000 x g for appropriate time (15 minutes for 22 uL volume)
- Discard flow through tube and get a new one
- Invert filter inside flow through tube and spin down at 14000xg for 1 minute
- Alternately, extract sample using pipette
Protocol-Dialysis[edit]
- Add X uL sample and new buffer to 500 uL total volume
- Spin down at 14000 x g for appropriate time (15 minutes for 22 uL volume)
- Discard flow through and add 500 uL new buffer
- Repeat steps 2 and 3 as many times as desired
Dialysis Table[edit]
Starting Sample Volume | 500 uL dilution | Original Buffer Concentration | Centrifuge Time |
50 | 10 | 10% | 15 min |
20 | 25 | 0.4% | 15 min |
20 | 25 | 0.016% | 15 min |