Re: [ccp4bb] Thin plate crystals

2015-04-29 Thread Phoebe Rice
Sometimes (sadly, not always) the problem with thin plates is that they get damaged in mounting. If none of the tricks suggested work, you might try seeing if you get any better data from the existing crystals using those tennis racket shaped loops that give more support to the thin plate.

Re: [ccp4bb] experimental phasing at low resolution

2015-04-29 Thread Phoebe Rice
Dear Bei, It can sometimes be difficult to tell a real molecular replacement solution from noise at low resolution. In addition to Eleanor's excellent advice, you might try to use your potential derivative data to test the top molrep solutions, even if their statistics are crappy. Try

Re: [ccp4bb] offtopic: AKTA prime

2012-07-13 Thread Phoebe Rice
If its old and out of warrenty, see if you have a local shop that can do it. We found the guys in physics here are great with such things (and a lot cheaper than official company repair guys). = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The

Re: [ccp4bb] The effect of His-tag location on crystallization

2012-06-27 Thread Phoebe Rice
With Flp recombinase - DNA complexes, a C-terminal His tag triggered a different (but sadly not better) crystal form, and the His side chains packed against the bases at the end of a neighboring DNA duplex. = Phoebe A. Rice Dept. of Biochemistry Molecular

Re: [ccp4bb] badly behaved DNA binder

2012-06-18 Thread Phoebe Rice
Try adding DNA then dialyzing to low salt (in some microdialyer). = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723

Re: [ccp4bb] resolution on PDB web page

2012-04-25 Thread Phoebe Rice
What freaked me out is that REMARK 2 seems to have changed over time: I have a version of 1ihf.pdb (deposited around 1995) that was apparently downloaded in 1998, where remark 2 says 2.5, and a version downloaded yesterday where remark 2 says 2.2. The whole thing actually started because the

[ccp4bb] resolution on PDB web page

2012-04-24 Thread Phoebe Rice
I just noticed that the PDB has changed the stated resolution for one of my old structures! It was refined against a very anisotropic data set that extended to 2.2 in the best direction only. When depositing I called the resolution 2.5 as a rough average of resolution in all 3 directions, but

Re: [ccp4bb] Crystal behave funny

2012-04-13 Thread Phoebe Rice
I'd suggest: - Find a dinosaur from my generation who can suck one into a capillary and check diffraction at room T. - Try using those loops that look like miniature tennis paddles to give the crystal a little more support - To minimize strain on the crystal when pulling it out of the drop,

Re: [ccp4bb] very informative - Trends in Data Fabrication

2012-04-02 Thread Phoebe Rice
Can we leverage this to push journals to routinely allow reviewers access coordinates and maps? Outright fraud is outrageous, but I'm actually more worried about ligands fit to marginal density and other issues of under-supervised model building. =

Re: [ccp4bb] very informative - Trends in Data Fabrication

2012-04-02 Thread Phoebe Rice
in a map calculated at a specific sigma and in different orientations. Maria On 2 April 2012 18:43, Phoebe Rice wrote: Can we leverage this to push journals to routinely allow reviewers access coordinates and maps? Outright fraud is outrageous, but I'm

Re: [ccp4bb] one datum many data? [was Re: [ccp4bb] very informative - Trends in Data Fabrication]

2012-04-01 Thread Phoebe Rice
Ah, an old pet peeve resurfaces! English is complicated and data is by now an English word. To use a somewhat strained analogy, at the quantum level, the word has a singular and a plural form, and at the classical-mechanics level, the word is a mass noun. Most crystallographers use the word

Re: [ccp4bb] Aggregated protein for crystallization

2012-02-21 Thread Phoebe Rice
I probably it depends on whether you've got gunk or a functionally relevant oligomer in that void volume. Is it active? RecA and Rad51 both form open-ended head-to-tail oligomers and yet they still crystallize. = Phoebe A. Rice Dept. of Biochemistry

Re: [ccp4bb] Reasoning for Rmeas or Rpim as Cutoff

2012-01-31 Thread Phoebe Rice
I'm enjoying this discussion. It also seems like a good spot to inject my standard plea for better treatment of anisotropy in things like table 1 of papers and PDB deposition forms. When you have Ewald's football (American football), like many nucleic acid-ophiles do, one number simply isn't

Re: [ccp4bb] Introducing an ELN

2012-01-26 Thread Phoebe Rice
As the proud owner of a carefully organized, highly annotated VMS backup tape (reel-to-reel, of course), my main concern is that paper is the only format that we'll be able to count on reading a decade (or more) from now. = Phoebe A. Rice Dept. of

Re: [ccp4bb] Expression of Viral proteins for crystallography

2012-01-25 Thread Phoebe Rice
We've seen nice in vivo activity (on purpose) from proteins cloned under T7 promoters but transformed into non-DE3 cells. In fact, friends working with more zesty enzymes who wanted a more tunable in vivo assay have had to mutate the ribosome binding sites for proteins under T7 promotors to

Re: [ccp4bb] Merging data collected at two different wavelength

2012-01-18 Thread Phoebe Rice
Can I be dogmatic about this ? I wish you could, but I don't think so, because even though those sources call it that, others don't. I agree with your thinking, but usage is usage. And 10,000 lemmings can't be wrong?

[ccp4bb] symmetry for ages 6 and up

2011-12-13 Thread Phoebe Rice
Hi all, For those who teach xtallography - we found some plastic turtles that can be snapped together in an amazing variety of space groups. Worked well in a workshop for our students, so I thought I'd share the shopping tip. They're called Reptangles, and we got them from Amazon.

Re: [ccp4bb] Protein-DNA complex crystallization

2011-11-21 Thread Phoebe Rice
What is Kd? Also, in reply to earlier posts: it is sadly common in crystallizing large protein-DNA complexes to go through a couple dozen different duplexes and several dismally-diffracting crystal forms before finding a good one. Phoebe From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on

Re: [ccp4bb] detect dsDNA

2011-10-01 Thread Phoebe Rice
Some things that are a good sign for having DNA as well as protein in your crystal: 1) You get different (or no) crystals if you add or substract a base or two from the DNA ends. Of course, we did get fooled by this logic once when one particular oligo seemed to be contaminated with an easily

Re: [ccp4bb] question regarding secondary-structure restraints

2011-09-22 Thread Phoebe Rice
Its also a bit too simple to count secondary structure restraints as 2 restraints per residue, because if they're tight enough on, say, an alpha helix, in combination with other geometry restraints (good bond angles, no clashes, etc) you could probably turn the backbone of the entire helix into

Re: [ccp4bb] Protein preps become a jelly

2011-09-16 Thread Phoebe Rice
Gamma delta resolvase catalytic domain stock solutions used to make a nice clear jelly at 4 degrees, but it was perfectly reversible by warming the sample to room T. In fact, one mutant crystallized in the stock tube after a few trips in and out of the fridge. The crystals didn't diffract

Re: [ccp4bb] refmac and DNA (and now RNA)

2011-09-12 Thread Phoebe Rice
Is there finally, at long last, one convention for nucleic acids? I wonder how many cumulative person-years of exasperation this @#$% issue has caused? And please note, even Mother Nature herself, let alone synthetic chemists, occassionally attaches U to deoxyribose or T to plain ribose.

Re: [ccp4bb] consistently missing eletron density

2011-08-12 Thread Phoebe Rice
beta sheets in really well-phased low-resolution maps should look sort of like walls, but in an imperfect map they might by rather spotty. I'd be very leary of making any conclusions from molecular replacement at such low resolution. For a good control, try solving your data set with a

Re: [ccp4bb] Fwd: Could Biological Negative Results be published?

2011-07-11 Thread Phoebe Rice
This brings up a philosophical caveat that one should be very careful about labeling one set of potential results negative and another positive before even doing the experiment, because its all too easy to see what you hoped to see even if it was only marginally there. The best

Re: [ccp4bb] low res. SAD phasing

2011-06-15 Thread Phoebe Rice
Based on the dents in my own forehead from banging it on similar brick walls, it would probably be more efficient to get a heavier atom with fewer binding sites, and work your way in from there. If your data set really only extends to 6A, even when you do find all those Se atoms their phasing

Re: [ccp4bb] DNA oligonucleotide purification

2011-04-28 Thread Phoebe Rice
We actually screen with unpurified oligos (up to just over 30nt) and it works just fine. Saves lots of time $$. If we get hopefull crystals, we pay for purification or do it ourselves by gel. Sometimes it makes the crystals better and sometimes it doesn't. Phoebe

Re: [ccp4bb] immobilized DNA resin

2011-04-11 Thread Phoebe Rice
As suggested, you can probably get good purification of heparin. If your pet protein has a known specific binding site, you can make it a personalized column by PCR'ing up arrays of directly repeated binding sites. The repeats will mis-anneal in subsequent rounds, giving rise to longer and

Re: [ccp4bb] Crystallographic Breakthrough - DarkMatter Version 1.0

2011-04-01 Thread Phoebe Rice
Congratulations on your amazing discovery, which immediately suggests many new lines of inquiry: Does dark matter affect macromolecular stability? Can it explain the difficulty some students have in sample preparation? Is it found in higher concentrations in brains that are thought to be

Re: [ccp4bb] what to do with disordered side chains

2011-03-31 Thread Phoebe Rice
- they all know what B is and how to look for regions of high B (with, say, pymol) and they know not to make firm conclusions about H-bonds to flaming red side chains. But this knowledge may be quite wrong. If the flaming red really indicates large vibrational motion then yes, one whould

Re: [ccp4bb] what to do with disordered side chains

2011-03-30 Thread Phoebe Rice
I've now polled 4 fairly savvy end users of crystal structures and there seems to be a consensus: - they all know what B is and how to look for regions of high B (with, say, pymol) and they know not to make firm conclusions about H-bonds to flaming red side chains. - None of them would ever

Re: [ccp4bb] while on the subject of stereo

2011-03-22 Thread Phoebe Rice
My 2 cents worth on the stereo-dependent: 1) They have carpal tunnel syndrome that makes it painful to keep the molecule in motion while rebuilding it (NOTE: enough constant mouse-wiggling and you will get carpal tunnel problems if you don't have them yet!) 2) They work on big, low-resolution

[ccp4bb] silly question on coot defaults

2011-03-20 Thread Phoebe Rice
I'm allergic to red nitrogens and blue oxygens, and I'm tired of changing the default way new files are colored every time I restart coot (I know it save the whole previous session, but sometimes I don't want to see that set of molecules again). I must be missing a way to save the preferred

Re: [ccp4bb] protein lost activity after size exclusion chromatography

2011-03-16 Thread Phoebe Rice
Depending on what the expected activity is, its worth considering the highly-depressing possibility that the activity seen in the impure sample was due to impurities: for example, barely-visible-on-a-gel chaperones can give a nice ATP hydrolysis signal, and DNA ligases float about with an AMP

Re: [ccp4bb] Noisy difference maps with high solvent content?

2011-01-29 Thread Phoebe Rice
You've probably done it already, but solvent flattening/flipping/massaging at 80% solvent should provide a cheap thrill with regard to phase improvement. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723

Re: [ccp4bb] Phase Separation

2011-01-13 Thread Phoebe Rice
It sounds a bit silly after that nice theoretical discussion, but I would try poking the existing oily blobs with a hair. Since you may be close to xtal conditions, stirring up the equilibrium a bit may help nucleate something. I've seen this work more than once, although usually with things

Re: [ccp4bb] SDS and IMAC

2011-01-03 Thread Phoebe Rice
Especially if you're dealing with lysate, I suspect the best way to do it is with magnetic Ni beads that you lift up and out of the gunk, to help avoid false positives from aggregating stuff that SDS/urea/guan would all elute. But why do you want X to remain on the column/beads? Removing Y but

Re: [ccp4bb] Citations in supplementary material

2010-11-22 Thread Phoebe Rice
I'm all in favor of (rational) length limits for the text - almost everybody's prose is easier to read when they've been forced to cut 10-20%. However, some journals include references in the character limit, and that just encourages unscholarly behavoir. Phoebe

Re: [ccp4bb] Citations in supplementary material

2010-11-17 Thread Phoebe Rice
Another unfortunate aspect of this sort of editorial policy is that many of these papers contain almost no technical information at all, except for the supplement. I've started to avoid using Nature papers for class discussions becuase they leave the students so puzzled, and with a

Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)

2010-10-27 Thread Phoebe Rice
Journal editors need to know when the reviewer they trusted is completely out to lunch. So please don't just silently knuckle under! It may make no difference for Nature, but my impression has been that rigorous journals like JMB do care about review quality. Phoebe

Re: [ccp4bb] Against Method (R)

2010-10-26 Thread Phoebe Rice
Another issue with these statistics is that the PDB insists on a single value of resolution no matter how anisotropic the data. Especially in the outermost bins, Rmerge could be ridiculously high simply because the data only exist in one out of 3 directions. Phoebe

Re: [ccp4bb] help with pymol error message

2010-10-19 Thread Phoebe Rice
I know people sometimes have good reasons, but people should be very careful about carving a map so close to the atoms - one could end up seriously misrepresenting the quality of the map. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The

Re: [ccp4bb] About SAD phasing

2010-08-30 Thread Phoebe Rice
Dear Qing, Many structures have been solved that way. Make sure you try solvent modification (flattening / flipping) on your map. This is because SAD will give two equally-probably estimates for the phase. The initial, unmodified map will use the average of the two, but solve modification

Re: [ccp4bb] Problems in purification

2010-08-26 Thread Phoebe Rice
Have you tried expression tricks like Rosetta cells? Testing different colonies and/or starting from fresh transformants? Sometimes that matters. If your protein is an oligomer and your contaminants are degradation products, you might try adding some urea. If desparate, you could spike the

Re: [ccp4bb] Extremely long c-axis...reasonable?

2010-08-03 Thread Phoebe Rice
There's no theoretical reason it can't be that long. BUT it is possible, especially for problematic diffraction patterns (e.g. from a badly-diffracting, cracked crystal) for the software to pick some wacky value in its attempt to fit spots that don't really all belong in the same pattern.

Re: [ccp4bb] Residual densities!

2010-07-21 Thread Phoebe Rice
Could any of those strong spots at 2.1 be ice? Original message Date: Tue, 20 Jul 2010 16:13:31 -0700 From: Lijun Liu Subject: Re: [ccp4bb] Residual densities! To: CCP4BB@JISCMAIL.AC.UK Pavel, Thanks for the quick response. I just listed out the Fo-Fc

Re: [ccp4bb] Introducing PDBprints - salient, at-a-glance info about PDB entries

2010-07-15 Thread Phoebe Rice
What would be wrong with WORDS? They were such a clever invention. I can tell the difference between colors, but it takes a second step to figure out what they mean anyway. Why not just write no info over the gray ones? And a 1-word caption on all the little icons would help, IMHO. Phoebe

Re: [ccp4bb] molecular replacement

2010-05-21 Thread Phoebe Rice
it it doesn't provide enough phasing power to crack the problem, you can use the positions of Se peaks in anomalous diff maps to check possible molecular replacement solutions. Good luck! Phoebe Rice Original message Date: Fri, 21 May 2010 20:23:23 +0530 From: Vineet Gaur vineetgaur1

Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?

2010-05-18 Thread Phoebe Rice
You could make use of product binding energy to drive the reaction forward while the substrate/product is bound to the enzyme. But enzymes that pull that trick are barely enzymes - they stay stuck to the first product they make until something else uses some energy to release it. You can't

Re: [ccp4bb] Re: [ccp4bb] Mysterious Crystals?

2010-04-19 Thread Phoebe Rice
Move the beam stop back? My lab has grown quite a few crystals that only diffract to very low resolution. Phoebe (with sympathy!) Original message Date: Mon, 19 Apr 2010 11:35:11 +0800 From: tat cheung cheng Subject: [ccp4bb] Re: [ccp4bb] Mysterious

Re: [ccp4bb] Need help for phasing using Tantalum bromide cluster

2010-03-05 Thread Phoebe Rice
We recently solved a multi-SeMet data set with nasty pseudotranslational symmetry by telling lies to the software: we (meaning my long-suffering student) indexed it in the smaller unit cell by picking only the dark spots, found the Se atoms in that cell, then reconstructed the larger cell

Re: [ccp4bb] UV microscope for screening

2010-01-20 Thread Phoebe Rice
Are these things really cost-effective? That is, compared to the cost of simply posting a list of evil salts like Mg ammonium phosphate on the wall and testing a few duds in the x-ray beam? Phoebe Original message Date: Wed, 20 Jan 2010 13:59:23 -0500 From: Gabriel Birrane

Re: [ccp4bb] FW: pdb-l: Retraction of 12 Structures....

2009-12-11 Thread Phoebe Rice
Yes, as much as I like to pick on the tabloids and their somewhat iffy quality control, it looks like this guy got away with faking structures for 10 years UNTIL he published in Nature. Even Acta Cryst has egg on its face. So the bottom line is that sunlight is indeed the best disinfectant, and

Re: [ccp4bb] molecular replacement in phaser

2009-11-10 Thread Phoebe Rice
We've had good luck (sometimes) searching for nucleic acids with multiple small pieces - say ask it to find several 5bp chunks of DNA rather than one 20bp duplex. I think Bill Scott will agree? I have a hunch that searching for the protein 1st, at least at modest resolutions, may confuse the

Re: [ccp4bb] I apologize

2009-08-21 Thread Phoebe Rice
And I apologize for being crabby about it. I realize its hard for people from different traditions and different native tongues to figure out the best way to start an English letter to people you've never met. Original message Date: Fri, 21 Aug 2009 16:50:28 - From: Debajyoti

Re: [ccp4bb] tips on crystallizing a Protein-DNA complex

2009-07-16 Thread Phoebe Rice
1. Do people routinely try different lengths of DNA? Yes, its usually the most important variable because the ends like to stack against each other to form a pseudo-continuous helix. 2. Do you start with blunt or sticky ends? Both. Plan on a dozen or so different duplexes to start

[ccp4bb] Best 3D stereo combination

2009-07-11 Thread Phoebe Rice
Sorry, still need help with this well-trodden territory: We're badly in need of a new linux PC with stereo and I'm confused about graphics cards. We want to recycle the CRT monitor, NuVision emitter and glasses from an old increasingly unstable machine. This chart:

[ccp4bb] Best 3D stereo combination for Linux

2009-07-01 Thread Phoebe Rice
Sorry this has probably come up already, but we're about to become desperate for a new stereo-capable machine, to be attached to an existing linux server. Is there a currently proven-to-work LCD stereo system for linux that works with Coot and/or O as well as pymol? Or should we recycle the

Re: [ccp4bb] Request recommendation for the peptide synthesis company

2009-06-02 Thread Phoebe Rice
For something that large, would it be easier and cheaper to make E. coli do it? Say, fuse your sequence to ubiquitin? Phoebe Original message Date: Mon, 1 Jun 2009 12:09:00 -0400 From: Pius Padayatti Subject: Re: [ccp4bb] Request recommendation for the peptide

Re: [ccp4bb] Phasing at Low Resolution

2009-05-14 Thread Phoebe Rice
My post-doc recently produced a splendid (for its resolution) ~5A map of a medium sized protein-DNA complex using Ta6Br12 clusters. And he's got a good toehold on a ~340kDa complex using the same clusters. So I'm recently converted to these little nuggets. Phoebe Original message

Re: [ccp4bb] Manipulating electron density

2009-05-11 Thread Phoebe Rice Subject: Re: [ccp4bb] Manipulating electron density To: CCP4BB@JISCMAIL.AC.UK On May 10, 2009, at 12:21 PM, Phoebe Rice wrote: Of COURSE the map will look lovely if you carve it off at 1.5A from your atoms. And your gels will look lovely too if you just touch them up

Re: [ccp4bb] Manipulating electron density

2009-05-10 Thread Phoebe Rice
Of COURSE the map will look lovely if you carve it off at 1.5A from your atoms. And your gels will look lovely too if you just touch them up with with some white-out and a sharpie. Do the honest thing and show the whole truth, using the z-clipping to get a comprehensible slab. Original

Re: [ccp4bb] two identical proteins in one asymmetric unit

2009-03-24 Thread Phoebe Rice
My favorite trick was to define domain-wise ncs restraints, extensively minimize with and without them, then plot the difference in real-space R factor per residue. Ones that jump up when restrained are usually involved in crystal packing, etc, and should be removed from the restraints. In my

Re: [ccp4bb] purification

2009-03-20 Thread Phoebe Rice
Try running the Ni column as fast as possible and putting concentrated EDTA in the fraction collector tubes before you start, to minimize opportunities for metal-dependent proteases. It may not be a magic bullet but it can't hurt. Phoebe Original message Date: Thu, 19 Mar 2009

Re: [ccp4bb] [SPAM:#] [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?

2008-12-11 Thread Phoebe Rice
Mass action is on the crystal's side. Two recent examples of proteins that are dimers by standard solution assays, but form weak/transient/co-factor-dependent tetramers to function, and those tetramers are seen in the crystal. (There is good solution data to back up the relevance of the tetramer

Re: [ccp4bb] MR with DNA

2008-12-08 Thread Phoebe Rice
There are certainly reasons why it shouldn't work: - the protein may alter the structure - even if its not a big bender, it may tweak the groove widths or introduce a shallow overall bend in a long binding site - the phosphates move the most between DNA structures, and unfortunately they also

Re: [ccp4bb] co-crystallization

2008-12-01 Thread Phoebe Rice
Just to be a pedantic pain - Km is not necessarily Kd. I think that assumption only holds if the chemical step following substrate binding is rate-limiting. Phoebe Original message Date: Mon, 1 Dec 2008 15:34:59 +0100 From: mesters [EMAIL PROTECTED] Subject: Re: [ccp4bb]

Re: [ccp4bb] Cryoprotectant for protein-DNA complex crystal

2008-11-03 Thread Phoebe Rice
I think we still have better luck with longer, slower, more gentle soaks - but its crystal-dependent. Try raising the [PEG] at the same time as you raise the [glycerol]. Phoebe Original message Date: Fri, 31 Oct 2008 18:57:53 -0400 From: Artem Evdokimov [EMAIL PROTECTED] Subject:

Re: [ccp4bb] Poor electron density - polyAla or PolyGly?

2008-10-22 Thread Phoebe Rice
I was always a big fan of lego loop in O for building loops into dotted lines. Phoebe Original message Date: Wed, 15 Oct 2008 16:20:26 +0100 From: Eleanor Dodson [EMAIL PROTECTED] Subject: Re: [ccp4bb] Poor electron density - polyAla or PolyGly? To: CCP4BB@JISCMAIL.AC.UK Joe

Re: [ccp4bb] Protein-DNA complex prepartion for crystallization

2008-10-06 Thread Phoebe Rice
Hi, Sadly, that happens sometimes. 1) make sure you have some salt in the DNA stock too 2) try (NH4)2SO4 instead of NaCl (just don't add Ca++, and remember the ionic strength of a given molarity will be higher) 3) if you have to, try more salt 3) try different ends on your oligos - sometimes

Re: [ccp4bb] truncate ignorance

2008-09-09 Thread Phoebe Rice
] *** - Original Message - From: Ethan Merritt [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, September 08, 2008 3:03 PM Subject: Re: [ccp4bb] truncate ignorance On Monday 08 September 2008 12:30:29 Phoebe Rice wrote: Dear Experts

Re: [ccp4bb] Disordered domains in crystal strutures

2008-06-30 Thread Phoebe Rice
in the rather lousy, low-resolution, anisotropic electron density maps. Phoebe Rice PA, Steitz TA. Model for a DNA-mediated synaptic complex suggested by crystal packing of gamma delta resolvase subunits. EMBO J. 1994 Apr 1;13(7):1514-24. Abdel-Meguid SS, Murthy HM, Steitz TA. Preliminary X

Re: [ccp4bb] query on DNA-protein complex preparation for crystallization

2008-06-23 Thread Phoebe Rice
You should add some salt when you anneal!!! The duplex is highly negatively charged, so adding even a small amount (like 10mM NaCl) will help with charge screening, thus making the two strands less repellant to each other. Buffer is also always a good idea. At low pH and high temp you'll

Re: [ccp4bb] crystallisation

2008-06-03 Thread Phoebe Rice
If you have high [DTT] in your buffer, you might be catalyzing the addition of dimethyl arsenic (from the cacodylate) to some of your cysteines? Also, 10% glyercol sounds quite low for reproducibly good freezing (at least in my experience). Phoebe Original message Date: Mon, 2 Jun