ListServ Archive: Drugs, Inhibitors, Reagents

Drugs, Inhibitors, Reagents

Reagents

Antibodies

Drugs and Inhibitors

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Reagents

Can anyone suggest a type of agarose to use that is comparable to agarose-M from LKB Pharmacia? That company is no longer in business and I cannot find that specific agarose. Is it alright for me to use a general agarose?
-Rachel S Hrynewycz, 8 Jun 2000

  • If you are looking at agarose to be used for the agar-overlay method, I have tested Bio-Rad's Biochemical Gradae Agarose and found works well for it. By the way, I got one from LKB Pharmacia a couple of years ago and recall that the Company had changed the Organization/Name. Try "Amersham Pharmacia Biotech, Inc," Piscataway, New Jersey.
    -Yoshio Fukui, Northwestern University, 8 Jun 2000

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Is DIF available for purchase from specific companies?
-Michael Myre, University of Toronto at Mississauga, Canada, 8 Oct 2001

  • DIF-1 can be obtained from: Affiniti Research Products Ltd, Mamhead castle, Mamhead, Exeter, EX6 8HD, England, Fax: +44 (0) 1626 891090, Tel: +44 (0) 1626 891010 www.affiniti-res.com. Best wishes,
    -Robert Kay, MRC Laboratory of Molecular Biology, England

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Antibodies

Does anyone know which company's anti-GFP antibodies worked well in co-IP experiment?
-Xuehua Xu, National Institute of Allergy and Infectious Diseases, MD, 24 Aug 2005

  • In my old lab in Paris I used (and they still use) the mouse polyclonal from Roche, it's a mixture of 3 monoclonals, a postdoc had compared all the existing anti-GFP at the time and found that it was the best one for IP.
    -Clément Nizak, Columbia U, NY

  • Our lab has been using a rabbit polyclonal GFP antibody from Molecular Probes and it has worked very well in co-IP experiments.
    Shawn Galdeen, Universit yof Minnesota

  • We use a polyclonal from BD: Full-length A.v. polyclonal Antibody (raised either against the full-length GFP) - Cat. No.632460.
    -Vassil Mihaylov, NIH, MD

  • I'm using Roche's anti-GFP to do co-IP right now. Actually it is a mixture of two mouse monoclonal antibodies, not three. This Roche anti-GFP works perfectly in western blot, very clean background with dicty lysate. In immunoprecipitation, I can capture the free GFP in cell lysate. I'm still working on my protocol to see whether it's able to pull down my GFP fusion protein. The initial trials failed. Generally, I'll recommend to give this anti-GFP a try.
    -Patrick Zhang, Baylor College of Medicine, TX

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Does anyone know of a good anti-tubulin antibody that works well in Dicty? There are lots on the market yet so far all the ones that I have looked at show no reactivity with Dicty.
-Keith Niven, Deakin University, Australia, 17 Oct 2005

  • The mouse anti tubulin monoclonal antibody works well in Dicty. I believe the monoclonal clone is DM1A or something like that.
    -Matt Kinseth, University of California, San Diego

  • I have also had good luck with this antibody [mouse tubulin antibody], especially labeling spindles.
    -Robert Pope, Indiana University South Bend, ID

  • The mouse anti tubulin monoclonal antibody works well in Dicty. I believe the monoclonal clone is DM1A or something like that.
    -Masazumi Sameshima, Hirosaki University, Japan

  • the YL1/2 antibody from Chemicon work great with any fixation. But be careful, it's a rat monoclonal, so you need anti-rat secondary antibodies. If you use high quality highly cross-adsorbed antibodies (e.g. from Jackson labs or Molecular Probes) you can even combine it with mouse antibodies.
    -Ralph Gräf, Ludwig-Maximilians-Universität München

  • 12G10 is a mouse monoclonal antibody that works very well in Dictyostelium. The antibody was generated against tubulin isolated from Tetrahymena. You can see 12G10 anti-tubulin immunofluorescence in Dicty in the publication Zhang et al., 2003. 12G10 monoclonal supernatant is available at cost from the Developmental Studies Hybridoma Bank at the University of Iowa (Google search "DSHB" for the catalog). DSHB routinely ships to clients worldwide so there is no problem sending the antibody to Germany. I hope this information helps you.
    -Karla Daniels, University of Iowa, IA

  • Our anti-tubulin protocols work quite well (Sigma = T6557, MAb GTU-88 ascites). The one we use for anti-alpha tubulin is also from Sigma = F2168, MAb DM1A clone alpha tubilin FITC. Sigma T9026 for the unconjugated.
    Protocols: Dictyostelium cell staining for tubulin, Dictyostelium anti-γ-tubulin Westerns.
    -Emma Dalton, Cardiff University, UK

  • I have used 12G10 and that worked beautifully in Dicty cells, You can get that form University of Iowa Hybridoma bank. Generated against Tetrahymena in Mouse. I have used as 1:150 dilution in PBS+2% BSA.
    -Taruna Khurana, NIH

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I am trying to do immunofluorescence studies with an anti-tubulin antibody in order to visualize microtubules. I was wondering which anti-tubulin antibody would be recommended, and what company I might be able to purchase it from.
-Catherine Socec, Vassar College, NY, 27 Jan 2005

  • Please send your Fed Ex mailing address, and I'll send you a couple of ml of our favorite monoclonal supernatent. It is a terrific antibody for Dicty MTs... The only string attached is that you should cite the original paper (Piperno and Fuller 1984. J. Cell Biology 101:2085-2094) for its production. Cheers,
    -Mike Koonce, Wadsworth Center, 27 Jan 2005

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I'm looking for antibodies against Dicty organelle markers (ER, Golgi, endosomes,...). The antibodies would be used for immunfluorescence and Western blotting.
-Dieter Klopfenstein, University of California San Francisco, 23 Jan 2001

  • Hi, Dieter (& others on the list): Do you know if there are any mito-specific antibodies? Thanks in advance.
    -Dale Hereld, University of Texas-Houston Medical School, TX, USA, 24 Jan 2001

  • Sure, there is a mitochondrial porin antibody mAb 70-100-1 that works in Western blots and immunofluorescence. Reference: Troll et al, 1992.
    -Guenther Gerisch, Max-Planck-Institut fuer Biochemie, Martinsried, Germany, 27 Jan 2001

  • Does anyone have a marker (either antibody or GFP fusion) that is specific for Golgi? Thanks. Best-
    -Rick Firtel, UCSD, CA, 13 Feb 2001

  • See Schneider et al 2000 'Golvesin-GFP fusions as distinct markers for Golgi and post-Golgi vesicles in Dictyostelium cells.'.
    -Margaret Clarke, OK Med. Res. Found., 13 Feb 2001

  • We have found fluorescent wheat germ agglutinin (Molecular Probes) to be a very convenient Golgi-selective stain.
    - Jeff Williams, Univ. of Dundee, UK

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I'm looking for a commercially available anti myc-antibody that works well with myc-tagged proteins in Dicty (primarily for westerns but maybe also for indirect immunofluorescence). Any suggestions? Thanks,
-Denis A. Larochelle, Clark University, Worcester, MA, 12 Sep 2000

  • Invitrogen
    -Rick Firtel, UCSD, CA 12 Sep 2000

  • We purchased the 9E10 cell line from ATCC and had a biotech company make a bunch of ascites. This antibody works great for westerns, indirect immunofluorescence, and IPs.
    -Elizabeth J. Luna, University of Massachusetts Medical Center, 12 Sep 2000

  • The 9E10 has worked well for us for Westerns and immunofluorescence. Good luck. regards,
    -Marcus Fechheimer, U. Georgia, 12 Sep 2000

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Drugs and Inhibitors

Does anyone know if aluminum fluoride or GTPγs were successfully used to mimic activation of Gα in living Dicty?
-Ewa Snaar-Jagalska, Univ. Leiden, The Netherlands, 9 Aug 2005

  • We showed many years ago that fluoride has dramatic effects on slug behaviour. It was simply incorporated into the agar. However we did not use the aluminium salt directly but used (if I remember correctly) KF. See Dohrmann et al. 1984.
    -Paul Fisher, La Trobe Univ., Australia

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Dear all, May anyone suggest me dose and time effect for Actinomycin D in Dd?
-Francesco Dondero, University of Piemonte Orientale, Aessandria, 9 Jun 2000

  • Check several J. Mol. Bio. papers in the early 1970's, Firtel and Lodish, maybe other authors as well. Best-
    -Rick Firtel, UCDS, 9 Jun 2000

  • See also Mizukami and Iwabuch 1970.
    -Masazumi Sameshima, The Tokyo Metropolitan Institute of Medical Science, Japan, 12 Jun 2000

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Inhibition of RNA synthesis: To those of you who are interested in inhibiting RNA synthesis and don't want to or can't use actinomycin, see Ennis, 1981 and Kelly et al., 1985. We used a compound called nogalamycin to inhibit RNA synthesis. It works well in Dicty, rapidly inhibits RNA synthesis and we used it to determine mRNA half-lives. The drug was made by Upjohn, Kalamazoo, MI.
-Herb Ennis, Columbia University, NY, 12 Jun 2000

  • As pointed out by Herb Ennis, nogalomycin is a useful inhibitor of RNA synthesis. Upjohn made a series of Nogalomycins (U12,241, U15,167 and U20,661) which we found to block spore germination. Other useful inhibitors reported to be RNA synthesis inhibitors which blocked spore germination included Daunomycin, Thiolutin and 4-nitroquinoline 1-oxide. We reported these results in Hamer, Cotter and Demsar, 1984.
    -Dave Cotter, U. Windsor, Canada, 12 Jun 2000

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I want to block protein synthesis in Dicty without affecting endocytosis and phagocytosis (especially in DH1 strain). Unfortunately, all the inhibitors that I thought to use (cycloheximide, puromycin, anisomycin, etc) are known to affect endocytosis and phagocytosis even at low dose. Does someone know another protein synthesis inhibitor that I can use which has less effect on endocytosis and phagocytosis? Maybe all the protein synthesis inhibitors have inhibitory effect on endocytosis and phagocytosis. If you have an idea, let me know... Thank you
-Steve Charette, Centre médical universitaire, Université de Genève, Switzerland, 1 May 2004

  • We used cycloheximide to block protein synthesis, leaving secretion intact, and it worked nicely (Monnat et al., 2000). The conditions are as follows: "Cells expressing the GFP constructs were treated for up to 4 h with 1.6 mM cycloheximide, a dose sufficient to block protein synthesis by 98% (Müller-Taubenberger et al, 1988)". Now you say that it blocks endocytosis, is this a reference to work published (I think I remember a report in a Dicty book by the Sartre/Grenoble people on something like that) or your own experience? [Note from Steve: Yes, it was the case. The paper is: Gonzalez C and Satre M 1991. Surprisingly this paper is not accessible trough PubMed or on the web site of JCS. I do not know why. I found the paper because it was referenced by another paper... and finally found the hard copy of the paper in the archive of library of my university!] If different inhibitors of protein synthesis with completely different modes of action block endocytosis, the simplest explanation is that a short lived protein is essential. I am interested in the response you will get to your question and to the results of such experiments. Best wishes,
    -Thierry Soldati, Imperial College London, UK

  • Years ago, we investigated several inhibitors of protein synthesis. In addition to cycloheximide and anisomycin, emetine was very effective, and also pactamycin. Emetine inhibition was readily reversible, without irreversible developmental effects; pactamycin, less so. Puromycin did NOT work for us on developing cells. I have not concerned myself with these drugs for a long while, and can't tell you now of other processes they may also target. Our reference, with concentrations and sources, etc., is Ratner, Pentz and Pelletier, 1989.
    -David Ratner, Amherst College, Amherst MA

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Has anybody used proteosome inhibitors in Dicty? Which ones and what concentrations did you find best?
-Jon Reynolds, 17 May 2002

  • There is some information in Schauer et al 1993 Proteasomes from Dictyostelium discoideum: Characterization of Structure and Function. Regards,
    -Guenther Gerisch, MPI f. Biochemie, Martinsried, Germany, 21 May 2002

  • We have been using MG132 at 20 µM, but do not see not obvious effects on the breakdown of cyclins, which in other organisms are typically destroyed by this pathway. All suggestions will be much appreciated!
    -Harry MacWilliams, Ludwig-Maximilians-Universität, Muenchen, Germany, 11 May 2003

  • We tried a number a few years ago on pathways that we knew were regulated by proteosomal degradation and saw not effect, including no build up of ubiquitinated products. I do not remember what we used but we also tried "industrial" strength levels. Since we tried a few, I am assuming that they either didn't get inn or, more probably, they got secreted rapidly by tyhe ABC transporters. Best-
    -Rick Firtel, UCSD, CA, 11 May 2003

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