[ccp4bb] hydrophilic protein going to aggregate

Hello Crystallographers, I am trying to express and purify a soluble domain of a membrane protein for crystallization. The amino acid content is as below Ala (A) 12 13.8% Arg (R) 10 11.5% Asn (N) 2 2.3% Asp (D) 8 9.2% Cys (C) 1 1.1% Gln (Q) 1 1.1% Glu (E) 4 4.6% Gly (G) 16 18.4% His (H) 3 3.4% Il

[ccp4bb] Guard columns from FPLC

Hi All, Sorry for this off topic. I have heard that there are these little columns called guard columns which can be attached to AKTA purifiers. These columns prevent the incoming huge aggregates to be deposited and blocking of the gel filtration columns. Can any one advice me regarding where to

Re: [ccp4bb] ITC with unfolded proteins

t Professor > The City College of New York > Department of Chemistry, MR-1135 > 160 Convent Avenue > New York, NY 10031 > Tel. (212) 650-6070 > > > Original message > >Date: Fri, 14 Mar 2014 18:07:48 +0530 > >From: CCP4 bulletin board (on behalf &g

[ccp4bb] ITC with unfolded proteins

Hello everyone, I have a query for the scientists working on protein-protein interaction. It is known that some proteins exist in unfolded or molten globule state and attain structure on interaction with other folded proteins. Many a times, it is difficult to obtain the structure of these complexe

Re: [ccp4bb] off topic: rmsf in simulation

ubstrate binding and activity. You might be interested in a review by >> Homans (ChemBioChem 6, 1585, 2005) which >> discusses the use of NMR to look at entropy changes in protein-ligand >> binding reactions. It is by no means unusual >> for a residue's entropy to increase

[ccp4bb] off topic: rmsf in simulation

Hi All, I am trying to understand the mechanism of protein-peptide interaction in two complexes (protein-pepA and protein-pepB). While trying to perform some simulation experiments, I find that the* root mean square fluctuation (RMSF) by residues of protein in the complex is higher than that of the

[ccp4bb] off topic: protein peptide binding

Hi All, I wanted some advice regarding mapping out Protein-peptide interaction. The peptide is a 12 mer and the protein is 15kDa. Invivo studies suggest that the peptide is binds the protein and helps in transport. Hence I feel it would perhaps transient binding. I know that I should do ITC or BIAc

[ccp4bb] generating symmetry related

Dear All, I have a small molecule structure file coordinates in CSD CIF format, I would like to analysis inter-molecular interaction between them by generating symmetry related nearest neighbor structures. I want to store the coordinates of the generated structures and further analysis it using in

[ccp4bb] off topic Thermal shift assay

Hi All, I want to use a thermofluor for the thermal shift assay. My proteins are cytoplasmic truncations of membrane protein. I have read about ANS, sypro-orange and CPM. Which is the once that is popularly used by the crystallographers for condition optimization for crystallization ?? I have read

[ccp4bb] detergent or protein

Hi All, I would like to have your expert advice on crystals. I am using detergents as 5% (w/v) of DDM 0.4ul in a 4ul (protein + condition + detergent). The precipitant is 28% peg 20K After 1 day I am able to see little plates of irregular shape . I am able to see some needles if I change into MEGA

[ccp4bb] granular precipitate

Hi All, I have set up initial screen in hanging drop trays with a protein of theoritical pI of 8.5. The protein is in acetate buffer 10mM, KCl 100mM and 2% glycerol pH 5 . In 85-90% of the conditions I see granular precipitate in 1 day. I tried to open the coverslip, and touch few drops, They had a

[ccp4bb] Na acetate as purification buffer

Hi all, Has anyone used sodium acetate buffer pH (4-5) for purifying histag protein on Histrap column (AKTA) followed by SEC? My protein has a pI of 9. I tried pH7.4 but it has precipitation problems. While doing buffer screening using 24 well hanging drop I found that lower pI onces are clear, so

[ccp4bb] native gels

Hi All, Has anyone run a native gel for proteins at pI>8 . I want to pour my own native gel. Do I run a discontinuous page or a continuous one?? Please help with regards to the buffer system to be used, and the dye to be used. With regards Rashmi

Re: [ccp4bb] drops swelling

such context: Vera,L., Czarny, B., Georgiadis, D., Dive, > V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. > Cryst. Growth & Des. 11 :2755–2762. " > > http://pubs.acs.org/doi/abs/**10.1021/cg101364m<http://pubs.acs.org/doi/abs/10.1021/cg101364

[ccp4bb] drops swelling

Dear Crystallographers, I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it. This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition.

Re: [ccp4bb] Protein aggregation and crystallization

ge - I would check it somehow, maybe by light > scattering or centrifugation > > Good luck > > Yury > -- > *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of anita p [ > crystals...@gmail.com] > *Sent:* Friday, August 26, 201

Re: [ccp4bb] Protein aggregation and crystallization

> detergent from the begining itself. > Please do not chop off the membrane part keep it > and chop some of the unstructured cytosolic part if you want. > all the best. > let us know if any of these worked > Padayatti > > On Fri, Aug 26, 2011 at 3:03 AM, anita p wrote: >

[ccp4bb] Protein aggregation and crystallization

Hi All, I am working on a protein which has a membrane spanning region and as cytosolic domain.I have made various deletion constructs of the protein, so that I can have a crystallizable fragment. There is no homologues mentioned in the pdb for this protein. All of these constructs are purified

[ccp4bb] detergents

Hi all, I have been trying to purify cytosolic fraction of membrane protein whose domain boundries are unknown. hence I have made a series of deletion constructs. The expression and purification is not a problem. I get good yields of the proteins. But on a gelfiltration column, they run in the void

[ccp4bb] reproducibility of protein crystals

Dear Crystallographers, I have got my protein crystallized once and then it is not reproducing, though I am using the same batch of protein and same condition. What are the reasons behind nonreproducibility of protein crystals? I am very new to this field hence I apologize if it is a lame questi

[ccp4bb] Detergents

Hi, I had set up crystallization with a bicine as buffer and peg 400 as precipitant. I used the detergent DDAO/LDAO as an additive to the crystallization drop (one of the hampton additive screen condition, it says 5% on the vial) I have a clear drop and in the centre there is a shiny precipitate (

Re: [ccp4bb] Tev Cleavage issue !!

phic separation e.g. ion exchange of HIC - they may or may not > work out. You can also consider cleaving your protein at lower > concentration, in the presence of detergents or polyols, etc. > > Cheers, > > Artem > > On Thu, Apr 7, 2011 at 9:37 PM, anita p wrote: > >&g

[ccp4bb] Tev Cleavage issue !!

Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. *Is it probable that they dont diffract because of the extra his tag and the tev site?* I am trying to get

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