fixed and DAPI stained therefore all fluorescence images were taken without

Fixed and dapi stained therefore all fluorescence

This preview shows page 11 - 13 out of 14 pages.

fixed and DAPI stained, therefore all fluorescence images were taken without fixing, precluding DAPI staining. Capsule blotting Estimation of shed capsule polysaccharide size and amount was performed using a technique described by Yoneda and Doering [47]. Conditioned media was made by growing strains in Dulbecco’s modified Eagle’s medium or low iron medium for 1 week at 30 u C with shaking. The cells were incubated at 70 u C for 15 minutes to denature enzymes, then pelleted for 3 minutes at 1500 rpm. The resulting supernatant was sterile filtered using a 0.2u filter. 15 uL of conditioned media was mixed with 6x load- ing dye and run on an agarose gel at 25V for 15 h. The polysaccharides were then transferred to a nylon membrane using Southern blotting techniques. The membrane was air-dried and blocked using Tris-Buffered Saline-Tween-20 (TBS-t) with 5% milk. To detect the polysaccharide, the membrane was incubated with monoclonal antibody 18b7 (1/1000 dilution) [87], washed with TBS-t, then incubated with an anti-mouse peroxidase- conjugated secondary antibody (1/25,000 dilution, Jackson Labs) C. neoformans Rim101 PLoS Pathogens | 11 February 2010 | Volume 6 | Issue 2 | e1000776
Image of page 11
and detected using SuperSignal West Fempto Maximum Sensi- tivity Substrate (ThermoScientific). Protein extraction, immunoprecipitation and western blot analysis Protein extracts were obtained using a method previously described [88]. Briefly, cells were incubated to an optical density at 600 nm of 1 in YPD and capsule-inducing conditions. Twenty- milliliter samples of growing cells were pelleted and resuspended in 0.5 mL of lysis buffer containing 2x protease inhibitors (Complete, Mini, EDTA-free; Roche) and 2x phosphatase inhibitors (Phos- Stop; Roche). To lyse the cells, the supernatant was removed and the cells were lysed by bead beating (0.5 mL of 3uM glass beads in a Mini-BeadBeater-16 (BioSpec), 4 cycles for 30 s each). Following lysis, the samples were immunoprecipitated using 1.6 m g anti-GFP antibody (Roche) for 1 hour, then rotated with 80 m L protein G Sepharose (Thermo Scientific) for 1 hour. After washing, the samples were eluted by the addition of 40 m L 5x Laemmli sample buffer and boiling. Western blots were performed as described previously, using NuPAGE Tris-Acetate gels or NuPAGE Bis-Tris gels to separate the samples. To detect the GFP-labeled proteins, the blots were incubated with anti-GFP primary antibody (1/5,000 dilution) and an anti-mouse peroxidase-conjugated secondary antibody (1/25,000 dilution, Jackson Labs). As a control for non- specific immunoprecipitation, samples were tested in a mock IP (in which no antibody was added) and probed with the anti-GFP antibody, and no bands were observed in this control experiment. RNA and cDNA preparation Strains were incubated to mid-logarithmic phase in YPD then washed three times with sterile water before incubation in capsule- inducing media for 3 hours. Cells were washed three times before centrifugation and freezing on dry ice and lyophilizing. RNA was prepared from lyophilized samples using the RNeasy kit (Qiagen).
Image of page 12
Image of page 13

You've reached the end of your free preview.

Want to read all 14 pages?

  • Fall '11
  • MarilynGadomski
  • DNA, C. neoformans

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern

Stuck? We have tutors online 24/7 who can help you get unstuck.
A+ icon
Ask Expert Tutors You can ask You can ask You can ask (will expire )
Answers in as fast as 15 minutes