ISISBio:Protocols/p11 resin preparation: Difference between revisions
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(New page: ==Overview== Preparation of Whatmann P11 Phosphocellulose resin for cation exchange chromatography. Phosphocellulose remains a useful and cheap cation exchange resin and is particularly...) |
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==Overview== | ==Overview== | ||
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# Suspend the resin in at least 20 volumes of water and allow to settle for at least 30 minutes | # Suspend the resin in at least 20 volumes of water and allow to settle for at least 30 minutes | ||
## Pour off supernatant, resuspend and repeat at least 8-10 times. | ## Pour off supernatant, resuspend and repeat at least 8-10 times. | ||
## | ##* This may appear appointless but it is crucial for getting rid of fine particles that will increase back pressure and reduce column performance. | ||
## | # Suspend the resin in 0.2 M NaOH,allow to settle for at least 30 minutes. | ||
## Pour of supernatant and repeat until pH of supernatant is higher than 10 | |||
# Suspend resin in water, allow to settle, pour off supernatant, and repeat until pH is seven or lower | |||
# Suspend resin in 0.2 M HCl, allow to settle, pour off supernatant, and repeat until pH is three or lower | |||
# Suspend resin in storage buffer, allow to settle, pour of supernatant, and repeat until pH is that of buffer | |||
#* Try to leave the resin at least once overnight in buffer at some point in this step | |||
# Suspend resin and pour carefully into column housing | |||
# If resin is to be stored, consider adding 0.2% sodium azide | |||
# Try to replace the buffer regularly as ammonia can be released from amine containing buffers | |||
==Notes== | ==Notes== | ||
# | # The repeated washing with water is critical to remove fines. Don't be tempted to shortcut this step! | ||
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | ||
==References== | ==References== | ||
<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. --> | <!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. --> | ||
==Contact== | ==Contact== | ||
* | * [[User:Cameron_Neylon|Cameron Neylon]] | ||
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | <!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | ||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:In vitro]] | |||
[[Category:Protein]] | |||
[[Category:Chromatography]] | |||
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[[Category:In vivo]] | [[Category:In vivo]] | ||
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[[Category:RNA]] | [[Category:RNA]] | ||
[[Category:Chemical]] | [[Category:Chemical]] | ||
Revision as of 04:11, 29 April 2008
Overview
Preparation of Whatmann P11 Phosphocellulose resin for cation exchange chromatography.
Phosphocellulose remains a useful and cheap cation exchange resin and is particularly good for the final stages of purification of DNA binding proteins. The preparation of the resin for optimum performance is somewhat tedious but carefull attention to detail will yield a higher performance coplumn with lower back pressure.
Materials
- Whatmann P11 Phosphocelluose resin
- Purified water
- 0.2 M NaOH
- 0.2 M HCl
- Storage buffer (Tris-HCl pH 7.6 is a common choice)
Procedure
- Weigh out approximately as many grams of resin as millilitres of column will be required into a large container with at least 20-fold larger volume
- Suspend the resin in at least 20 volumes of water and allow to settle for at least 30 minutes
- Pour off supernatant, resuspend and repeat at least 8-10 times.
- This may appear appointless but it is crucial for getting rid of fine particles that will increase back pressure and reduce column performance.
- Pour off supernatant, resuspend and repeat at least 8-10 times.
- Suspend the resin in 0.2 M NaOH,allow to settle for at least 30 minutes.
- Pour of supernatant and repeat until pH of supernatant is higher than 10
- Suspend resin in water, allow to settle, pour off supernatant, and repeat until pH is seven or lower
- Suspend resin in 0.2 M HCl, allow to settle, pour off supernatant, and repeat until pH is three or lower
- Suspend resin in storage buffer, allow to settle, pour of supernatant, and repeat until pH is that of buffer
- Try to leave the resin at least once overnight in buffer at some point in this step
- Suspend resin and pour carefully into column housing
- If resin is to be stored, consider adding 0.2% sodium azide
- Try to replace the buffer regularly as ammonia can be released from amine containing buffers
Notes
- The repeated washing with water is critical to remove fines. Don't be tempted to shortcut this step!
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
