Re: [ccp4bb] DDM
It is important to distinguish between the solubilisation and the purification steps: 1) During the solubisation step you need to care about the lipid/detergent ratio. The amount (not the concentration) of detergent (i.e. in non monomeric form, the detergent above the cmc) is important. You may need a high amount of detergent. 2) During the purification step, you need to keep your protein soluble. Here, the concentration is important and you may keep the concentration of detergent around the cmc. But you have to be aware that in the initial (and also the not so initial ones!) steps you may have a lot of lipids, you need then keep the concentration of detergent fairly high in order to keep everything soluble. I like to decrease the detergent concentration at each purification steps in order to avoid protein denaturation. The protein may sustain a fairly high detergent concentration of detergent during the early steps since the lipid/detergent mixed micelles will be less denaturing than the pure detergent micelles in the later steps. Thin layer chromatography is a quick and easy method to check if you have a large amount of lipids in your preparation. HTH Daniel Le 26/03/2012 19:17, Katarzyna Rudzka a écrit : Hi All, Has anyone had any luck purifying membrane proteins with DDM (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC is very low: 0.009%). I would like to keep it as low as possible, so I don't have too much DDM around when I get to the crystallization step. I wonder If the amount of detergent sufficient for the protein extraction has to be determined experimentally for each protein or maybe there are some good rules of thumb. I appreciate your help. Thanks. Kasia Katarzyna Rudzka, Postdoctoral Fellow Department of Biophysics and Biophysical Chemistry Johns Hopkins University, School of Medicine Baltimore, Maryland 21205 USA
Re: [ccp4bb] DDM
Actually, DDM is the most successfully used detergent for membrane protein crystallization. See Newstead et al, Protein Sci. 17:466. But yes, the rule of thumb is that detergents that form smaller micelles give better diffracting crystals, but are more destabilizing. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu On Mon, Mar 26, 2012 at 1:06 PM, CCP4BB automatic digest system wrote: > Date: Mon, 26 Mar 2012 17:39:57 + > From: Joao Dias > Subject: Re: DDM > > Kasia, > A lot of people uses DDM to purify membrane proteins, not a lot of people > crystallises them. > If you want to crystallise a protein purified in DDM, then you should use LCP. > If you go for vapour diffusion, you should exchange the DDM for a detergent > with a smaller micelle size otherwise you might get crystals but it is > difficult to get good diffraction. Try mixed micelles for example. > Typically use 0.05% DDM during purification and use 100kDa cut-off membranes > in order to prevent detergent concentration. > For extraction it depends on your protein and expression system but you can > see in the literature values between 0.5-2% being used successfully. > Good luck. > Cheers, > Joao > > Joao Dias, Ph.D. > > Senior Scientist > Heptares Therapeutics Ltd > BioPark, Broadwater Road, > Welwyn Garden City, > Herts, AL7 3AX > UK
Re: [ccp4bb] DDM
If I recall correctly cytochrome oxidase, which I believe was the first protein purified with DDM, requires about 10x cmc in column buffers to keep it soluble. Check for papers from 1970's or 80's by S. Ferguson-Miller. Cytochrme bc1 complex, on the other hand, is perfectly clear in 1 cmc and can be diluted from that into detergent-free buffer for spectroscopywithout becoming turbid, at least within an huor. My rule of thumb for solubilizing with DDM is 1 g/g protein (+ 1 cmc). If protein is say 10-20 g./l, the (+ 1 cmc) becomes completely insignificant. (Total protein, not the protein of interest. Probably depends on the amount of lipid too, but this is just a rule of thumb to start with.) eab Katarzyna Rudzka wrote: Hi All, Has anyone had any luck purifying membrane proteins with DDM (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC is very low: 0.009%). I would like to keep it as low as possible, so I don't have too much DDM around when I get to the crystallization step. I wonder If the amount of detergent sufficient for the protein extraction has to be determined experimentally for each protein or maybe there are some good rules of thumb. I appreciate your help. Thanks. Kasia Katarzyna Rudzka, Postdoctoral Fellow Department of Biophysics and Biophysical Chemistry Johns Hopkins University, School of Medicine Baltimore, Maryland 21205 USA
Re: [ccp4bb] DDM
Kasia, A lot of people uses DDM to purify membrane proteins, not a lot of people crystallises them. If you want to crystallise a protein purified in DDM, then you should use LCP. If you go for vapour diffusion, you should exchange the DDM for a detergent with a smaller micelle size otherwise you might get crystals but it is difficult to get good diffraction. Try mixed micelles for example. Typically use 0.05% DDM during purification and use 100kDa cut-off membranes in order to prevent detergent concentration. For extraction it depends on your protein and expression system but you can see in the literature values between 0.5-2% being used successfully. Good luck. Cheers, Joao Joao Dias, Ph.D. Senior Scientist Heptares Therapeutics Ltd BioPark, Broadwater Road, Welwyn Garden City, Herts, AL7 3AX UK This document contains company confidential and/or proprietary information. It is intended for the exclusive attention of the addressee(s) above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender. [cid:image6c7082.GIF@43484746.4ab9695e]<http://www.heptares.com> The information in this e-mail is confidential and may be legally privileged. It is intended for the exclusive attention of the addressee stated above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender. Contact and registered office address: Heptares Therapeutics Limited, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AX. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katarzyna Rudzka Sent: 26 March 2012 18:17 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DDM Hi All, Has anyone had any luck purifying membrane proteins with DDM (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC is very low: 0.009%). I would like to keep it as low as possible, so I don't have too much DDM around when I get to the crystallization step. I wonder If the amount of detergent sufficient for the protein extraction has to be determined experimentally for each protein or maybe there are some good rules of thumb. I appreciate your help. Thanks. Kasia Katarzyna Rudzka, Postdoctoral Fellow Department of Biophysics and Biophysical Chemistry Johns Hopkins University, School of Medicine Baltimore, Maryland 21205 USA <>
Re: [ccp4bb] DDM
Hi Katarzyna, Yes, membrane proteins can be purified in DDM at ~1-2xCMC. Since its CMC value is very low, at the extraction step you need to use a much higher concentration up to ~100x CMC. During different rounds of purification, you can bring the CMC level down to 1-2x CMC and even try detergent exchange in the last step, although it can be difficult to totally exchange out the DDM due to its low CMC. Also use a 100 kDa MWCO concentrator since the DDM micelle size is large. In general, DDM is a reasonable choice for extraction, purification and crystallization setups, but it will be worthwhile to screen a panel of different detergents for extraction and purification for your particular target. In 2005, we set up a fast protocol for screening a panel of 18 detergents in 48-72 hours, http://smb.slac.stanford.edu/~debanu/posters/012605_PPCW2005_DDAS.pdf, which includes suggestions for starting concentrations for extraction. There are also some papers on detergent screening, for example: http://www.ncbi.nlm.nih.gov/pubmed/18988031 http://www.nature.com/nprot/journal/v4/n5/full/nprot.2009.27.html (Stroud lab) Thanks, Debanu. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katarzyna Rudzka Sent: Monday, March 26, 2012 10:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DDM Hi All, Has anyone had any luck purifying membrane proteins with DDM (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC is very low: 0.009%). I would like to keep it as low as possible, so I don't have too much DDM around when I get to the crystallization step. I wonder If the amount of detergent sufficient for the protein extraction has to be determined experimentally for each protein or maybe there are some good rules of thumb. I appreciate your help. Thanks. Kasia Katarzyna Rudzka, Postdoctoral Fellow Department of Biophysics and Biophysical Chemistry Johns Hopkins University, School of Medicine Baltimore, Maryland 21205 USA
Re: [ccp4bb] DDM
I generally use 1 - 2% DDM for extraction only, but lower the concentration to 0.01% for following steps (i.e. NiNTA and gel filtration). The excess DDM is washed away by using a lower concentration in your wash and elution buffers. Kelly *** Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *** On Mon, Mar 26, 2012 at 1:17 PM, Katarzyna Rudzka wrote: > Hi All, > Has anyone had any luck purifying membrane proteins with DDM > (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its > CMC is very low: 0.009%). I would like to keep it as low as possible, so I > don't have too much DDM around when I get to the crystallization step. I > wonder If the amount of detergent sufficient for the protein extraction has > to be determined experimentally for each protein or maybe there are some > good rules of thumb. I appreciate your help. Thanks. > Kasia > > > Katarzyna Rudzka, Postdoctoral Fellow > Department of Biophysics and Biophysical Chemistry > Johns Hopkins University, School of Medicine > Baltimore, Maryland 21205 USA >
Re: [ccp4bb] DDM
Hi, I used 0.017% or 0.012%. My protein is very stable at this concentration. Good luck. Lin 2012-03-26 yybbll Hi All, Has anyone had any luck purifying membrane proteins with DDM (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC is very low: 0.009%). I would like to keep it as low as possible, so I don't have too much DDM around when I get to the crystallization step. I wonder If the amount of detergent sufficient for the protein extraction has to be determined experimentally for each protein or maybe there are some good rules of thumb. I appreciate your help. Thanks. Kasia Katarzyna Rudzka, Postdoctoral Fellow Department of Biophysics and Biophysical Chemistry Johns Hopkins University, School of Medicine Baltimore, Maryland 21205 USA
[ccp4bb] DDM
Hi All, Has anyone had any luck purifying membrane proteins with DDM (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC is very low: 0.009%). I would like to keep it as low as possible, so I don't have too much DDM around when I get to the crystallization step. I wonder If the amount of detergent sufficient for the protein extraction has to be determined experimentally for each protein or maybe there are some good rules of thumb. I appreciate your help. Thanks. Kasia Katarzyna Rudzka, Postdoctoral Fellow Department of Biophysics and Biophysical Chemistry Johns Hopkins University, School of Medicine Baltimore, Maryland 21205 USA
Re: [ccp4bb] DDM crystals
What exactly is your question--I saw tons of "crystals" of DDM and PEGs, I think especially P400, if I recall correctly. JPK On Wed, Jan 11, 2012 at 7:12 AM, Patrick Loll wrote: > Does anyone have any experience with formation of crystals of dodecyl > maltoside in the presence of PEG? > Pat > > --- > > Patrick J. Loll, Ph. D. > > Professor of Biochemistry & Molecular Biology > > Director, Biochemistry Graduate Program > > Drexel University College of Medicine > > Room 10-102 New College Building > > 245 N. 15th St., Mailstop 497 > > Philadelphia, PA 19102-1192 USA > > > (215) 762-7706 > > pat.l...@drexelmed.edu > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] DDM crystals
Does anyone have any experience with formation of crystals of dodecyl maltoside in the presence of PEG? Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry & Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] DDM artifacts?
> ps. in passing, it seems like it would be a great idea to get together an > excel database of all false-positive results (e.g. phosphate salt crystals) > commonly found in the usual crystal screens. One could then search it to see > whether one's current crystallization conditions have be villified in the > past. Good idea - I've set up a page on CCP4 wiki to record such occurrences: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Conditions_prone_to_salt_cyrstallization In theory, none of the screens shall produce salt crystals unless a) there is something in your protein buffer or b) drops dried out. So report you protein buffer content and don't report cases of salt crystals in 3 years old trays. -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] DDM artifacts?
Jacob,
I doubt that the spherulites formed in the presence of detergents are
"primary" detergent phenomenon. However, detergent micelles and the
protein-detergent aggregates (please don't say protein-detergent
micelles) will condense (phase) out in the presence of high salt or
polymers to form droplets. What you have to remember about these
droplets is (1) the membrane protein concentration is markedly
increased (up to 100-200 mg/mL or into the mM range) and (2) the
detergent concentration also increases (10-100x).
The first situation with a membrane protein can lead to spontaneous,
but uncontrolled nucleation that can lead to microcrystal or
paracrystal formation of the protein. Hence, spherulites form from
the droplets.
In the second situation, the detergent concentration can also cause
the system to reach one of the more ordered mesophases. While for
most nonionic and zwitterionic detergents, the concentrations we use
are well below these mesophases in all cases. However, we mix these
detergents with all sorts of other components (salts, PEGs, MPD,
etc.) which can markedly alter the phase behavior of the detergent.
Thus, droplets of highly concentrated detergent in the presence of
crystallization reagents could be induced to form liquid crystals,
which leads to spherulite formation.
As virtually all of the published phase diagrams for our detergents
of interest are of binary (detergent and water) and ternary
(detergent, low salt, and water) systems, they are useless for
predicting what is happening. Moreover, there is the dreaded specter
of contamination of your detergent. For example, alpha-octyl
glucoside crystallizes quite easily, while the beta anomer does not
under almost all conditions. Although most commercial sources of
beta-octyl glucoside are pretty pure (~98-99% pure), the detergent-
rich droplets could be enriched in the unwanted alpha contaminant.
Similar paracrystalline structures can be seen with contaminants of
other nonionic and zwitterionic detergents.
My advice to you is to see if the same phenomenon occurs in protein-
free controls drops. Remember that your membrane protein is a
"perturbant" of detergent phase behavior, and the protein-detergent
aggregate will also have its own phase behavior. If spherulite
formation ONLY occurs in the presence of your membrane protein, then
you could argue that the spherulites are made up of poorly
crystallizing membrane protein. Not completely a positive result,
but it is something.
Sadly, there are no papers about this. However, the Crystals page of
the Kay Diederichs' CCP4wiki (http://strucbio.biologie.uni-
konstanz.de/ccp4wiki/index.php/Crystals) now has a section for hints,
tips, & observations about crystals ("I have crystals, but are they
salt?"). I would encourage people to post their insights there.
Michael
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334Email: [EMAIL PROTECTED]
On May 22, 2008, at 8:58 PM, Jacob Keller wrote:
Sorry for this non-CCP4 question, but I know no better venue to ask
this:
Do people see detergent spherulites or other artifacts in crystal
screens in the presence of dodecyl-maltoside or other detergents?
Are there any papers about this? I have seen some papers talking
about the relationships between salt, temp, cmc, and cloud points,
but nothing on precisely this topic (detergent-related
crystallization artifacts).
Best Regards,
Jacob Keller
ps. in passing, it seems like it would be a great idea to get
together an excel database of all false-positive results (e.g.
phosphate salt crystals) commonly found in the usual crystal
screens. One could then search it to see whether one's current
crystallization conditions have be villified in the past.
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***
[ccp4bb] DDM artifacts?
Sorry for this non-CCP4 question, but I know no better venue to ask this: Do people see detergent spherulites or other artifacts in crystal screens in the presence of dodecyl-maltoside or other detergents? Are there any papers about this? I have seen some papers talking about the relationships between salt, temp, cmc, and cloud points, but nothing on precisely this topic (detergent-related crystallization artifacts). Best Regards, Jacob Keller ps. in passing, it seems like it would be a great idea to get together an excel database of all false-positive results (e.g. phosphate salt crystals) commonly found in the usual crystal screens. One could then search it to see whether one's current crystallization conditions have be villified in the past. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] ***
