Re: [ccp4bb] suitable buffer for CD studies
Harsh, This article describes common buffers for CD on page 2: http://www.ncbi.nlm.nih.gov/pubmed/17406547 Or this article on page 8: http://www.ncbi.nlm.nih.gov/pubmed/16027053 It seems like phosphate is the best, because it has low absorption at 180-200nm region. From organic buffers Tris/H2SO4 seems to the the best. As I remember, Cl absorbs a lot in that UV region. Vitali On Wed, Mar 20, 2013 at 1:59 AM, Harsh Bansia spideysp...@gmail.com wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh
[ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
Hi, Sorry for off-topic question. Does anyone have experience of the stabilisation of water-soluble proteins by detergents? Protein I'm working with is definitely water-soluble and has high yield, but, unfortunately, not very stable. Especially during concentration. So, we thought that adding some detergents may one of the ways to stabilise protein. So, did anyone do it before or may be know published examples? Any suggestions on the detergent type/concentration would be welcome. Thanks, Vitali
Re: [ccp4bb] Poor baculovirus stability at 4C?
Alexander, We produce baculovirus at Gibco SF900-II + 10% heated FBS + 1xPSG and then store at 4C routinely. And it seems to be quite stable. We have a virus that had no change for more than 2 years: we judge by the volume of it needed to infect_cells/produce_protein, no titer measurements. We also avoid exposure to light: definitely cover it with foil if it's transparent door fridge. Vitali On Thu, Sep 27, 2012 at 5:42 PM, aaleshin aales...@burnham.org wrote: Sorry for an off-topic question. 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 media switch, or something else? What is the best way to store baculovirus? Thank you Alexander Aleshin Sanford-Burnham Medical Research Institute Infectious Inflammatory Disease Center 10901 North Torrey Pines Road La Jolla, California 92037
Re: [ccp4bb] off topic: reduced glutathione interfering with protein activity?
Peter, Yes, in our hands it can eluted completely. Usually 20 ml of elution buffer per 2 ml of GS4B resin (GE healthcare, fast flow) is enough . We use 30 ml open-columns for purification - each column has 2 ml of resin. Vitali On Wed, Aug 29, 2012 at 3:02 PM, hsuu...@u.washington.edu wrote: Hi Vitali, I usually prep my GST elution buffers fresh, and make sure to pH it after dissolving the powder. So I don't think that's the problem, but good to know that you can use as low as 5mM GSH. Do you know if you knock off most of the protein from the resin at that concentration of GSH? Thanks, Peter On Wed, 29 Aug 2012, Vitali Stanevich wrote: Peter, Such high concentration of GSH may change the pH according to our experience. We usually use 50 mM Tris pH=8.0, 5 mM GSH, 3 mM DTT - so that you can load the sample on ion-exchange column after elution. If your protein is not stable without NaCl - you should add it also. Vitali On Wed, Aug 29, 2012 at 12:42 PM, Peter Hsu hsuu...@u.washington.edu wrote: I don't think so since I purify in the presence of reducing agents (DTT/BME) and I got activity out of the prep that was released by on column cleavage. On the other hand, I don't usually add those fresh each time I use the buffers so it's entirely possible the reducing agents are nowhere near the initial reduced form they were in initially. On Wed, 29 Aug 2012, Antony Oliver wrote: GSH will reduce your protein quite nicely - is your enzyme activity redox sensitive? --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On 8/29/12 5:56 PM, Peter Hsu hsuu...@u.washington.edu wrote: Hi all, I've been purifying my protein off a GST column and have noticed a massive difference in activity of my protein between a prep that was freed from the column via on column cleavage, and a prep that was eluted (20mM GSH) and then cleaved and further purified. I'm suspecting that the glutathione is somehow modifying/inhibiting my protein in some way, despite having removed the glutathione from the buffer via dialysis/ion exchange. I don't see anything out of the ordinary in my electron density that would suggest that glutathione has affected my protein in some way, but the huge difference seen in my activity assay suggests otherwise. My question is, has anyone else seen an effect from glutathione affecting their protein in some way? My second question is, what's the minimum amount of glutathione necessary to elute your protein from a column? Sorry for the off topic question and thanks for any responses, Peter
Re: [ccp4bb] Off-topic His-Antibody
The one we have is rubbish. I can tell a vendor name if you want off-list. A lot of non-specific bands + high background level makes it almost impossible to use against cell lysate. It sort of works against purified His-tag protein, but not perfect either. On Mon, Jun 25, 2012 at 4:33 PM, D Bonsor dbon...@ihv.umaryland.edu wrote: Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish.
Re: [ccp4bb] Off topic about application of detergent on non-membrane protein crystallization
Donghui, There is detergent screen from Hampton: http://hamptonresearch.com/product_detail.aspx?cid=1sid=39pid=31 We set up detergent screen for crystal conditions we'd like to improve: 200nl(buffer) + 200nl(protein) + 40nl(detergent). Occasionally (very occasionally) it works, but don't put to much hope on it. Vitali On Thu, Jun 14, 2012 at 9:35 PM, wu donghui wdh0...@gmail.com wrote: Dear all, I wonder if anyone has successful experience on using detergent to crystallize non-membrane protein. Based on what criteria to choose detergent to help solubilize and crystallize your non-membrane protein. Thanks for any input or comments. Best regards, Donghui
Re: [ccp4bb] SUMO(ULP-1) protease
Gloria, Why don't you introduce TEV-cleavage site? You can clone it between His- and sumo-protein_of_interest (if you want co-crystallise sumo with your protein) or between His-sumo and protein_of_interest (if you want to crystallise your protein only). Vitali On Thu, May 24, 2012 at 2:41 PM, Gloria Borgstahl gborgst...@gmail.comwrote: My fellow crystallographers, We are thinking the SUMO/His vectors would be nice to have in the lab aresenal... but. The stumbling block is that the protease needed for cleavage is very expensive at crystallography scale. SUMO(ULP-1) protease costs ~$700/mg fusion protein. It would not be a problem for labs using micrograms of protein, but is prohibitive at our level of protein purification. Has anyone found a way around this? Your pal, Gloria
Re: [ccp4bb] Sequence Alignment Question
You can try this site: http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi If you have .pdb for one of your sequences, you'll be able to show secondary structure also. vitali On Thu, Aug 18, 2011 at 6:31 PM, Yuri yuri.pom...@ufl.edu wrote: Hello Everyone, A little off topic but, what is a good way to show (publication quality) multiple sequence alignment? I am trying to show conserved regions in related proteins from different organisms. Thank you -- Yuri Pompeu
