Re: [ccp4bb] anaerobic crystallization
Chris Although generally less popular nowadays, microbatch-under-oil has great advantages for anaerobic work. You can keep your stock solutions in the chamber all the time, so you only need to degas your protein. Microbatch finds roughly as many crystals in screening experiments as vapor diffusion (summary at http://www.douglas.co.uk/mbnvdall.htm ). Of course the oil helps to protect the protein too - generally less is denatured on the surface For an automatic setup that fits in an anaerobic chamber conveniently see the winning entry in Douglas Instrument's competition (2006), from the University of Georgia, Athens http://www.douglas.co.uk/presentations/winner2_files/v3_document.htm Hope that's useful Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Christopher Rife Sent: 02 September 2009 18:34 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] anaerobic crystallization Hi, Does anyone have tips or suggestions for getting started with anaerobic crystallization? Searching through the archives, I was able to find some discussion about various glove box options (http://tinyurl.com/ccp4-glovebox), but not about the process itself (we already have a box). To simplify things, would it be worthwhile to perform the initial screens in something like the pre-filled Qiagen screens (http://tinyurl.com/qiagen-xseal)? Do I need to worry about evaporation while solutions are being degassed (esp volatiles)? Better/easier to try microbatch? Thanks for any input. Chris ___ _ ___ This is an e-mail from Danisco and may contain confidential information. If you are not the intended recipient and you receive this e-mail by mistake, you are not allowed to use the information, to copy it or distribute it further. Please notify us and return it to Danisco by e-mail and delete all attachments. Thank you for your assistance. ___ _ __
Re: [ccp4bb] anaerobic crystallization
Dear Chris, The answer depends on why you want to exclude oxygen for crystallisation and how many screens you want to do. Did you purify your protein in the absence of oxygen? If you only want to set up a few screens for a single construct - I would suggest you put the screens and the plates in your anaerobic chamber a week before you want to use them. Then you set up the drops manually either in linbro plates or in 96 stripwell plates that you can seal 8 reservoirs at a time. Depending on the size of your anaerobic chamber you can also put a microscope in there to look at the drops (otherwise I would suggest you get plexiglass boxes that you can close tight to take the plates out for inspection). I have never tried but it should also be posssible to pipett the drops with an 8-channel pipett with some practice. You could prefill and seal the plates outside bring them into the chamber let them equilibrate for a week and then set up the drops. I personally think that the Quiagen plates are quite large and you usually don't have too much storage space in the anaerobic chamber. Best regards, Alexander -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Christopher Rife Sent: Wednesday, September 02, 2009 7:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] anaerobic crystallization Hi, Does anyone have tips or suggestions for getting started with anaerobic crystallization? Searching through the archives, I was able to find some discussion about various glove box options (http://tinyurl.com/ccp4-glovebox), but not about the process itself (we already have a box). To simplify things, would it be worthwhile to perform the initial screens in something like the pre-filled Qiagen screens (http://tinyurl.com/qiagen-xseal)? Do I need to worry about evaporation while solutions are being degassed (esp volatiles)? Better/easier to try microbatch? Thanks for any input. Chris ___ This is an e-mail from Danisco and may contain confidential information. If you are not the intended recipient and you receive this e-mail by mistake, you are not allowed to use the information, to copy it or distribute it further. Please notify us and return it to Danisco by e-mail and delete all attachments. Thank you for your assistance. __
Re: [ccp4bb] anaerobic crystallization
Hey there Chris, Additionally, I have seen trays set up under Argon atmosphere in glove bags. They are sealed and left in the bag until inspection where they are temporarily brought out and returned. Then, when harvesting, they would use a glass dish (taller than the tray plus some working area) on the microscope and continually fill/purge with argon. Interestingly, as dense as argon is, it will displace nearly all naturally present gases as it falls to the bottom of the dish and pushes everything else up and out. This was to exclude oxygen, and did according to the structure. In the inorganic lab, they use what is known as freeze-pump-thaw(-repeat). To de-gas even volatile solvents, place your solvent/solution on a Schlenk line...freeze to liquid nitrogen temperatures...pump using (high) vacuum...thaw...replace with inert gas (nitrogen)...repeat. After the last pump, thaw without replacing nitrogen. Then, wire the Schlenk tube/flask shut, and send into the vacuum chamber of your drybox. It is best to take the solvents in independently, and then make up your reservoirs as there is a very small loss of some solvents. Kris - Kris F. Tesh, Ph D Director, Macromolecular Products Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA 001 281 362 2300 x 144 From: alexander.schif...@sanofi-aventis.com To: CCP4BB@JISCMAIL.AC.UK Date: 09/03/2009 03:06 AM Subject: Re: [ccp4bb] anaerobic crystallization Dear Chris, The answer depends on why you want to exclude oxygen for crystallisation and how many screens you want to do. Did you purify your protein in the absence of oxygen? If you only want to set up a few screens for a single construct - I would suggest you put the screens and the plates in your anaerobic chamber a week before you want to use them. Then you set up the drops manually either in linbro plates or in 96 stripwell plates that you can seal 8 reservoirs at a time. Depending on the size of your anaerobic chamber you can also put a microscope in there to look at the drops (otherwise I would suggest you get plexiglass boxes that you can close tight to take the plates out for inspection). I have never tried but it should also be posssible to pipett the drops with an 8-channel pipett with some practice. You could prefill and seal the plates outside bring them into the chamber let them equilibrate for a week and then set up the drops. I personally think that the Quiagen plates are quite large and you usually don't have too much storage space in the anaerobic chamber. Best regards, Alexander -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Christopher Rife Sent: Wednesday, September 02, 2009 7:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] anaerobic crystallization Hi, Does anyone have tips or suggestions for getting started with anaerobic crystallization? Searching through the archives, I was able to find some discussion about various glove box options (http://tinyurl.com/ccp4-glovebox), but not about the process itself (we already have a box). To simplify things, would it be worthwhile to perform the initial screens in something like the pre-filled Qiagen screens (http://tinyurl.com/qiagen-xseal)? Do I need to worry about evaporation while solutions are being degassed (esp volatiles)? Better/easier to try microbatch? Thanks for any input. Chris ___ This is an e-mail from Danisco and may contain confidential information. If you are not the intended recipient and you receive this e-mail by mistake, you are not allowed to use the information, to copy it or distribute it further. Please notify us and return it to Danisco by e-mail and delete all attachments. Thank you for your assistance. __
