Re: [ccp4bb] Resuspension of bacterial cell pellets

Roger Rowlett wrote: No air in the vessel, no foam. What manufacturer/model do you use? I can't quite imagine a beater that would have no air in the chamber but maybe there is something new under the sun. Yield of soluble, active protein from broken cells is quite comparable or better th

Re: [ccp4bb] Resuspension of bacterial cell pellets

Roger Rowlett wrote: This goes straight into a bead beater for complete, gentle homogenization in 8 min. Didn't you mean "complete, foam-producing, surface denaturation-inducing" homogenization? I am not saying that bead beater is worse than the "locally near boiling temperatures-producing"

Re: [ccp4bb] Poor baculovirus stability at 4C?

We began experiencing a sudden reduction in stability of baculovirus stock stored at 4C, like the titer drops more than 5 fold in 3-5 month. The only difference compared with previous preparations is a switch from Gibco SF900-II to a media from Lonza (Insect-XPRESS). Could it be due to the me

Re: [ccp4bb] co-express two proteins in E.coli

I have been using the Duet system from Novagen (or whatever it is called these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of my proteins did not work in either vector. Either, one protein expressed or the other. I played around with the promotors (they are both T7) by chang

Re: [ccp4bb] offtopic: packing gel filtration columns

Sorry for the slew of offtopic posts, but does anyone here have any experience repacking the large 120mL Superdex75/200 columns? Any advice/tips on doing it? I've got an older column that's gotten clogged while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and not sur

Re: [ccp4bb] Akta vs HPLC

What makes the Aktas different from HPLCs? Nothing. Akta Purifyer *is* HPLC. Dima

Re: [ccp4bb] Off-topic-Cleavage with TEV protease

I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT? Yes, no problem. TEV is slowly inactivated oxidation of the active site cysteine but that's about it. If you absolutely must have no reducer during cleavage, simply up

[ccp4bb] A little perspective (Re: Trends in Data Fabrication)

Just came across it and thought it's quite relevant with regard to the latest furor over fraud in crystallography: http://www.nature.com/nature/journal/v483/n7391/pdf/483531a.pdf "53 papers were deemed 'landmark' studies ... scientific findings were confirmed in only 6 (11%) cases". Mean num

Re: [ccp4bb] Desalting columns

I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions

Re: [ccp4bb] about point mutation

Phusion requires that the primers are phosphorylated for mutagensis to work, unlike Pfu. If you cannot phosphorylate them use Pfu as recommended by Charlotte Not really. Phusion *protocol* requires phosphorylated primers but seemingly the only reason for that is that they needed to find a way

Re: [ccp4bb] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]

I can observe the amplified PCR product before and after DpnI digestion (see image in http://ompldr.org/vY2x3aw), but cannot get any colony on LB plates. I'm using very fresh super competent cells so that I've got dozens of colonies with 60 ng of the parental/non-mutated vector as positive contr

Re: [ccp4bb] Refmac and metal on a two-fold?

I've seen this happening to water molecules as well (in a somewhat unpredictable fashion). In the latest refmac versions, you can try harmonic restraints, although these will only slow down the atom drift, as the target position is updated every cycle. Perhaps you can use distance restraints aga

[ccp4bb] Refmac and metal on a two-fold?

Hello, I have a metal ion sitting on a two-fold. I assigned it an occupancy of 0.5 but Refmac keeps refining it away from that position so that in the end there is a symmetry-related ion 1.6A away. I thought that this problem has been rectified long ago? Certainly I had several occasions where

[ccp4bb] Summary: sealing slides on VDX plates

Thanks to everyone who replied! This is a summary in case anyone is wondering about the same: 1. A clear majority of replies was along the lines of "just buy pre-greased plates". 2. Next in popularity was self-greasing with Dow Corning high vacuum grease. 3. Several replies suggested mixing

[ccp4bb] sealing slides on VDX plates?

Hello, I wonder what everyone is using for sealing hanging drop slides on VDX plates? For the most part, we paraffin oil but I am unhappy with it because it is too think and too frequently there is break in the oil and the drop dries too much. I find vacuum grease to be not terribly practical

Re: [ccp4bb] Akta Prime / FPLC Options / Off Topic

As a personal touch, I also find Biorad software much more intuitive. This discussion, of course, is similar to comparing car brands - experiences differ. What mystifies me is a need for a service maintenance contract at all. Has hardware become so much less reliable than in the past? Grante

Re: [ccp4bb] detect dsDNA

>> There exists a less toxic chemical than EtBr to stain DNA: SYBR safe >> DNA stain (a fluorescence dye sold by a certain vendor). > > SYBR Safe is about 10X less sensitive though. Can you do the toothbrush test with SYBR Safe? I wouldn't do that. As it is considerably more hydrophobic, I'd ex

Re: [ccp4bb] detect dsDNA

There exists a less toxic chemical than EtBr to stain DNA: SYBR safe DNA stain (a fluorescence dye sold by a certain vendor). SYBR Safe is about 10X less sensitive though. I suspect that not many chemicals in the lab are less toxic/mutagenic than EthBr. The classic Ames test shows that 5 ug of

Re: [ccp4bb] is codon optimization worth it?

In theory, if the rare codons are all covered by Rosetta's extra tRNAs, codon optimization should not make any difference. In practice it does because frequently it's not codon optimization per se but changing local mRNA structure. - Dima

Re: [ccp4bb] Linux vs MacOS for crystallographic software

Simon Kolstoe wrote: Meanwhile I think windows is slowly improving as a crystallography platform - and Microsoft is perhaps no longer hated in principle - however the one student in our lab who opted to go the windows route seems very limited in the software he can run. I have a feeling that

Re: [ccp4bb] Silver staining Coomassie stained gels

Can anyone suggest me a protocol for silver-staining the PAGE that is already stained with Coomassie. There is absolutely nothing wrong with silver staining of a Coomassie-stained gel without destaining. It only prevents "blowout" of most intense bands already well-visible with Coomassie. The

Re: [ccp4bb] TCA or acetone precipitation of proteins

Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye wit

[ccp4bb] Summary: N-terminal sequencing facility?

Thanks so much to everyone who replied! Just in case anyone needs a similar info in the future, here are the responses that I got: *** Peter Cherepanov: we had good experience with Pick 'n Post (Alphalyse: ht

[ccp4bb] N-terminal sequencing facility?

Hello, Can anyone recommend a facility that does N-terminal protein sequencing very well? It has to be able to work with PVDF-blotted protein bands as a source. Relatively inexpensive would be a plus. Thanks! - Dima

Re: [ccp4bb] Off topic - transformation problems

In this case, pGEX4T3 vector also expressed inserts constitutively. http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low leak expression from PTAC promoter. Definitely low

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

The method is that by Edelhoch, mentioned a couple of times already in this discussion. You recommended "determining extinction coefficients experimentally". How is plugging number of specific residues into a formula constitute experimental determination? It's also described in the paper by

Re: [ccp4bb] methods to capture proteins from cell culture medium

I thought about HIC too, but do not know if it would work since the binding of protein to HIC need high salt conc. and I am not sure if the salt conc. in the sf900 or Hi5 medium is high enough (the formulation is secret, LOL), thus it is good to know that someone has succesful experience with

Re: [ccp4bb] immobilized DNA resin

heparin sulfate has the highest negative charge density of any known biological molecule. Seems to me that phytic acid (IP6, C6H6-(H2P04)6) and inositol heptakisphosphate (IP7) should have higher charge per mass and per volume. - Dima

Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue

It is not surprising that your bradford and BCA assays don't agree if you have no aromatic amino acids in your protein. Bradford dye binds to hydrophobic residues, mainly aromatics, so I would guess your bradford is consistantly giving lower measurements than the BCA assay. I also wouldn't be s

Re: [ccp4bb] S-200 buffer-based peak shift?

At 07:23 PM 3/22/2011, Jacob Keller wrote: Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which wo

Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments

they buy it from some small company, idea being the applicability to SLS, optimized for minimized bleeding/sheding or material from the column, which will show up only in the light scattering detector. I don't know. Every single spec of Wyatts columns looks exactly the same as "Bio SEC-5" serie

Re: [ccp4bb] E. coli mutant strains

> It surely is not. An N-end rule has to do with ubiquitination, and it is > absent in E.coli. Not true. There is indeed and N-end rule in prokaryotes, including E. coli. Mediated by the ClpP protease-based system. See: http://www.cell.com/molecular-cell/abstract/S1097-2765%2808%2900692-8 Ooop

Re: [ccp4bb] E. coli mutant strains

Recently I am expressing one protein in BL21(DE3) and the protein undergoes N-terminal degradation. I am trying to keep this crucial N-terminal tail on the protein, which has MRS at the first 3 positions. Digging in to the literatures, I found the N-end rule, which tell t

Re: [ccp4bb] Density sharpening with Truncate?

At 05:39 PM 2/25/2011, Pete Meyer wrote: Or could anyone suggest a program that would be of help? CAD scaling with a scale factor of 1.0 and negative B-factor (isotropic or anisotropic) should do the trick. I haven't had much luck with density sharpening (at least at ~4-5 Angstroms), but oth

Re: [ccp4bb] off-topic: tag removal

Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. 2009 Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119 Unless the protein in question happen to be Leu as a C-terminal residue, this

Re: [ccp4bb] off-topic: tag removal

I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease whi

Re: [ccp4bb] Could someone can help me to explain why EDTA-2Na can formate salt crystals

No, there are not any other cations, so I feel very strange. Everything brought from sigma. Nothing's strange. EDTA is very poorly soluble at pH 4.2 and would become even less soluble in the presence of 47% PEG. So it crystallizes. Dima On Tue, Feb 22, 2011 at 8:58 PM, William Scott <

Re: [ccp4bb] SDS and IMAC

In my experience, either urea or guanidinium crashes out in gels. I can't remember--which one is it? I am thinking guanidinium. (If the answer to this email saves one grad student from the aggravation of such a phenomenon, it will have been worth it...) It's GuHCl and what crashes is dodecyl su

Re: [ccp4bb] Mg2+ or water

Sorry, the attachment is in here. Doesn't look like Mg2+ at all. Distances are too long, Mg is never coordinated by amides and if it were Mg you would have seen waters around it. Looks like tightly bound water to me. - Dima On Mon, Dec 20, 2010 at 4:16 PM, jlliu liu <

Re: [ccp4bb] hydrohyapatite column

I was wondering if any of you would be kind enough to share her/his experience with me, and would suggest vendors and models for such columns. I really like "ceramic" hydroxyapatite from Bio-Rad. The only type that behaves in the columns long-term, without the need for repacking. It also gives

Re: [ccp4bb] Crystal gel band

I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a

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

What about the possibility of double-blind review? I have actually wondered why the reviewers should be given the author info--does that determine the quality of the work? Am I missing some obvious reason why reviewers should know who the authors are? I've always felt (and advocated long time ag

Re: [ccp4bb] Quality of His-Select Resin After Regeneration

The Agarose superflow material is I think the same for Talon (Clontech) and Qiagen, not sure about Machery & Nagel or Sigma's product. Superflow agarose is cross-linked differently than "normal" sepharose/cross-linked agarose. The nature of chelates and their attachments also differ. Qiagen, S

Re: [ccp4bb] Off-topic: crystallization after refolding

However, treatment with urea is known to modify proteins (N-term/Lys/Arg), which could ultimately effect crystallization. Is this something that people generally worry about? For example -would you bother cleaning up the urea by ion exchange -get ultra pure urea (ultra $$$) -change to guanidin

Re: [ccp4bb] insect cell media

We have happily made a transition last year from using Invitrogen's SFM medium and cellfectin to Insect-Xpress (Lonza) and polyethyleneimine for transfection. We are moving several protein targets to large-scale cultures and would consider cost-cutting alternatives. For example, Invitrogen and The

Re: [ccp4bb] Phosphatase, which is the best?

I need to dephosphorilate blunt ends and, surfing on internet I found this two enzymes: - Shrimp Alkaline Phosphatase (SAP) - Calf Intestinal Alkaline Phosphatase (CIP) Have you any experience about these phosphatases, I mean wich is the best for blunt ends? Either will do dephosphorylation p

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

It could also be that the high impact factor of these journals, and their 'tabloid' character ensures that they are read by more people than other journals. So any bad data or fraud that gets published in Nature, Cell or Science is more likely to get noticed and talked about, than something that a

Re: [ccp4bb] Solubilization buffer

We use 8M urea solubilization buffer for our protein in inclusion bodies and recommended temperature is 10-15C. but in 8M conc the urea does not dissolve and is in crystalline form only, will it have any effect on solubilzation efficiency. Our solubilization time is 1 Hr and after that we centr

Re: [ccp4bb] mammalian cell culture on IMAC

When mammalian cell culture is being loaded to GE HisTrap resin Ni ions are being stripped off the resin, at least in my hands. Did any of you have similar experience and if so what kind of work-around was found? Volume is fairly large (3L) and concentration/dialysis have proven to cause loss

Re: [ccp4bb] Lipid Removal from Proteins

I dug around on the net and found this method to remove lipids from proteins: More precisely, from denatured proteins. That's what methanol/chloroform phase does for most proteins. "Wessel & Fluegge (1984), Anal. Biochem. 138:141-143. Itґs a methanol/ chloroform precipitation and gives you a

Re: [ccp4bb] DNA binding protein

I had a simple question about DNA binding protein. Is there an easy way to detect if your heterologously expressed protein is bound to DNA post purification. Yes. UV absorbance. DNA absorbs UV strongly, proteins do not. DNA absorbs 260 more thn 280, the opposite is true for proteins. I

Re: [ccp4bb] Best 3D stereo combination

This chart: http://www.nvidia.com/object/IO_11761.html combined with the CCP4 wiki's advice (that the card should say "stereo" under display connectors) imply that we need to spend at least $760 on the video card alone. Is that really true??? EEK!! Not nesessarily. Most GeForce cards can be s

Re: [ccp4bb] TEV nucleotude sequence with restriction site

Does anybody have a TEV-protease-site-coding nucleotide sequence with a commonly-used restriction site in it, preferably right at the end? Alternatively, does some somebody know of a program to determine all equivalent codon permutations for a small coding region, filtered for resulting restric

Re: [ccp4bb] issues with TLS refinement

The issue is: While running tls and restrained refinement, the program doesn't perform the given no. of tls refinements. For e.g., if you ask it to perform 10 TLS and 5 restrained refinement rounds, it'll run may be 2 or 3 rounds of TLS and then jump to restrained refinement rounds and finish

Re: [ccp4bb] CCP4BB Digest - 4 May 2009 to 5 May 2009 (#2009-124)

I incubated my pelets in 8M urea for a couple of hrs at 37C, spun and collected the supernatant, and then refolded the protein over a Ni column, as I had a his tag protein. To do this, you load and wash the protein normally, but before eluting, run b-cyclodextrin (i think 0.1%, you'll have to t

Re: [ccp4bb] IPTG induced protein expression

I was wondering if anyone has used regular IPTG (not IPTG-dioxane free, special grade) for protein expression. Are there any problems using such regular samples (5mg dioxane per Kg of IPTG) for protein expression? I have. No problem whatsoever. I belive the whole dioxane-free thing is about

Re: [ccp4bb] Off-topic: chemical modification on thiol groups

I am looking for a reagent (and vendor) that will irreversible put a raher bulky substituent on a free SH group and that does not react with free amines (or other potential reactive groups present on a protein surface). The connection with crystallography is that it is required for an experimen

Re: [ccp4bb] Replacement for arp_waters?

Donnie Berkholz wrote: One of the major reasons our lab started using Coot instead of O is that you didn't have to memorize or look up a million obscure functions to do everything. It was in the Coot GUI. When we're training new people, this helps immensely for the 99% of them who have never us

[ccp4bb] Correction: Refmac and MSE?

In the previous message I forgot to mention: When MET were modelled, there were no difference peaks anywhere. When I changed them all to MSE, the large difference density peaks showed up. Large *negative* difference density.

[ccp4bb] Refmac and MSE?

Hello, I am at a loss on what's going on: I am refining SeMET containing structure and using REFMAC 5.2.0005 on Linux and, the same thing happening, using REFMAC 5.5.0070 on Windows. When MET were modelled, there were no difference peaks anywhere. When I changed them all to MSE, the large di

Re: [ccp4bb] 3D modeling program

> But how do we establish phylogeny? - Based on simple similarity! > (Structural/morphological in early days and largely on sequence > identity today). It's clearly a circular logic: Hardly. Two sequences can be similar and non-homologous at all levels. Also, two similar proteins can be homologo

Re: [ccp4bb] 3D modeling program

But how do we establish phylogeny? - Based on simple similarity! ah! the old rhetorical trick of changing the problem or question a posteriori! all i pointed out was that things can't be "25% homologous" Well, you were right that in today's definition things can't be. But you seem to be miss

Re: [ccp4bb] 3D modeling program

Having a generic dictionary definition is nice and dandy. However, in the present context, the term 'homology' has a much more specific meaning: it pertains to the having (or not) of a common ancestor. Thus, it is a binary concept. (*) But how do we establish phylogeny? - Based on simple simil

Re: [ccp4bb] 3D modeling program

to models built on low-homology structures. since i'm currently preparing my bioinformatics lectures for next week's teaching, i might as well be a Besserwisser and point out that homology, much like pregnancy and death, is a binary concept. i'm sure artem knows this and simply mistyped "low

Re: [ccp4bb] Quikchange cloning: Insert length

I am curious to hear what is the longest insert anyone has cloned using a modification of the Quikchange cloning strategy. Basically, ligation-independent cloning by strapping on homologous regions of the vector onto the primers which also generate the initial PCR product. I plan to proceed with m

Re: [ccp4bb] purification of phosphorylated proteins-off topic

I am trying to find a method to purify/separate phosphorylated protein from unpshosphorylated proteins. I have two approach in my mind * ion-exchange * Fe columns, I tried this too but limited success. Gadolinuim-charged IDA with a shallow gradient of imidazole is supposed to separate ph

Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags

Another theory is that trace amounts of Ni may leech off the column during purification and coordinate with multiple His-tags on the pure protein, causing them to aggregate. For sure. We had a case where a protein would precipitate after post-Ni-NTA dialysis. The precipitate would go back into

Re: [ccp4bb] off-topic: selective reduction of surface cysteine

We have a recombinant secreted glycoprotein produced in a mammalian culture system; the native protein has 12 cysteines which form 6 intramolecular disulfide bonds. We have introduced a new cysteine residue at a surface position, with the intention of targeting this residue for an in vitro sit

Re: [ccp4bb] regarding cloning

1. Typical [restriction based] sub-cloning 2. Go through (TOPO)-TA cloning 3. Gateway cloning 4. LIC, Ligation independent cloning 5. SLIC, Sequence and Ligation independent Cloning. At the risk of being repetitive (since I posted it in a previous "cloning" thread), there is another very attrac

Re: [ccp4bb] AW: [ccp4bb] hi / cloning stuff

I found out that my glycerol stocks were not alright any more and I had no plasmid backups (do it!!!). So I had to clone, again. Hint for the future: Dead glycerol stocks, even dead (say, a year old) clone on a plate, still contain your plasmid. Do a regular miniprep with it, transform - 99% o

Re: [ccp4bb] agarose-acrylamide composite gels

Sorry for the off-topic question. I am trying to separate by SDS-PAGE really big proteins (>500 kDa), and lower percentage (3-8%) acrylamide gels do not do the trick. Based on literature searches, acrylamide-agarose composite gels seem the way to go. Is anyone willing to share a protocol? I can

Re: [ccp4bb] Expression vector with NdeI-ClaI sites

...Or do the slightly-less-new-but-not-quite-old school technique: QuickChange. Find a vector with roughly the properties you want, and just quickchange mutagenize in NdeI and ClaI sites at the desired positions. Works like a charm for me, assuming the vector is <10Kb or so, and I typically bu

Re: [ccp4bb] Concentrating protein

Nathan Cowieson: I have found that ion exchange is a good way to concentrate protein. If there is no reason that you can't pass the whole 450 mls through the column then you should do that. Another way is to load dilute protein onto small hydroxylapatite column. Provided that the buffer does

Re: [ccp4bb] Structural importance of ordered water?

Direct hydrogen bonds between sidechains are obviously important to structural stability in proteins. From time to time I see cases of water-mediated bonds in which a single water molecule seems to play an important role (sometimes taking the place of a missing ligand atom in an apo structure, for

Re: [ccp4bb] 3D model building without CRT monitor

Vangelis Christodoulou wrote: Philips is offering an interesting solution: http://www.business-sites.philips.com/3dsolutions/products/3dscreens/index.html Interesting! Their PDF says it works with OpenGL: http://w

Re: [ccp4bb] insect expression system

I searched my mail box for possible answers you have given but couldn't find any. The question is about selecting insect expression systems for mammalian or viral glycosylated proteins. There is confirmed expression of the target proteins in mammalian cells. They are secreted into the media.

Re: [ccp4bb] Off topic: PDI overexpression in HEK293 cells

With regard to low levels of expression: Is there a particular reason to use 293 cells? Transient expression in Cos-1/Cos-7 cells gives ~ 10 fold higher levels of expression because of the plasmid amplification in these cells. (The only requirement is that the plasmid should have SV40 ori; most ma

Re: [ccp4bb] Copper Staining

There's an even faster reagent for staining - takes abut 1 minute overall and the results look just like dear old Coomassie. This reagent works wonderfully for us. Yes, the recipe is proprietary - but if you add up the cost of reagents for normal Coomassie and compare - this one isn't much more ex

Re: [ccp4bb] extra line of stain in our gel

When we develop our gel we recently keep getting a horizontal line of stain at 5 mol weight in all lanes of our gel. (This is not a feature of interest except that the protein that we are interested happens to be 5 mol weight). I would appreciate any ideas on how we can get rid of

Re: [ccp4bb] Question about protein expression (not CCP4 related)

1. I was wondering if anyone used the "Multisystem Expression" vectors from Novagen and if so whats your opinion about these vectors? These are the pTriEx series of vectors that can be used for protein expression in E.coli, baculovirus and mammalian cells using the same construct. Since most

Re: [ccp4bb] Is phophorylation possible in E. coli expression system?

However, if your protein is not a protein tyrosine kinase, you may check your western condition. Yes, this is an essential control. Use lots of Lambda protein phosphatase to "dephosporylate" your protein and us the resulting material as a negative control in Western. My experience is that pho

Re: [ccp4bb] The importance of USING our validation tools

I like to emphasize that the infamous table 1 alone should have immediately tipped off any competent reviewer. The last shell I/Isig is 1.3 and rmerge 0.11 (!). And keep in mind that this statistics comes from merging data from FOUR different crystals! (That's clearly and unambigously stated in

Re: [ccp4bb] DNase inhibitors

I have small crystals of protein DNA complex. I am worried about the possibility of presence of some DNases in the drop (I use DNase in the lysis buffer). I have Tris and MgCl2 in the crystallization condition, which makes me hesitant to use DEPC or EDTA/EGTA respectively. Are there any other D

Re: [ccp4bb] CCP4 GUI

The level of detail in the GUI is a matter of constant debate. The underlying programs are far far richer, so the question is how much to expose in the GUI. How about the simple and elegant way it is done in Refmac? Tacked under "Developers Options" is "specify an external keyword script file fo

[ccp4bb] Hints on oil phase in the presence precipitants?

Hello, I've got a protein that forms oil phase in almost all conditions in the screen. No xtals thus far. I was wondering if there are general hints on dealing with such cases. Basically, I am trying to understand what direction to go from here. E.g. decrease or increase [protein], [salt] and

Re: [ccp4bb] Expression hosts (was:Inclusion body)

As has been suggested, you can try other hosts - for instance Gram positive bacteria (Bacillus megaterium is an interesting host as are other bacilli, especially if your protein is secreted), other Gram negative bacteria (Pseudomonas for instance), Can anyone comment on availability of Archaea e

Re: [ccp4bb] Bio-rad DuoFlow

(FPLC is a trademark. The marketing hype has been so effective that many people think that FPLC is a method.) Well, it is, in a way. The FPLC thingie was a pure marketing genius that bundled the remarkable technology of Monobeads (AFAIK, there is still no other company that offers comparable s