Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/2 June 2015"

(Created page with "{{Template_All_Teams}} <!-- Declare that you are going to use html code instead of wiki code --> <html> <!-- Start of CSS--> <style type="text/css"> →‎PAGE LAYOUT: ...")
 
 
Line 311: Line 311:
  
 
** Flourescence following washes of Tamura.
 
** Flourescence following washes of Tamura.
 +
 +
** SElective binding to silk sequence regions.
 +
 +
** Expectation of 100mg per 1L of cell culture.
 +
 +
*** 1ng minimum per well for measureflourescnece. 
 +
 +
**** Tamura - 00.5% of total silk, 2%,
 +
0.5% is 25ug per well, 1% 50 ug per well
 +
2% - 100 ug per well. 
 +
 +
**** Net STEP: Make hydrogels in PBS tomorrow.

Latest revision as of 00:02, 3 June 2015

  • Experimental Design Brainstorm
    • Tamura-GFP Hydrogel assay
      • Negative control - sfGFP without Tammy.
      • Experimental protein - Tamura
      • Assay for binding potential to doped silk solution.
        • Used stripping buffers to assay for agitation. (detergents, alkalinity, ionic strength, pH).
      • Differing variables: Concentration of Dope, concentration of Tamura in hydrogel.
      • Temperature based gradient on hydrogel stability.
      • Assay for flourescence using microplane reading, distribution using fluorescence intensity (read into measuring this quality).

Characterization of Tamura -

    • SDS-PAGe gels, Western Blot to assay degradation.
    • CD, IR Spectrum, Fluorescence Intensity of purified protein and compare that with protein concentration - relative to that metric (ug).
    • Cry - EM for silk Tamura fibers - make sure that we don't mess with the fibers.
    • Gel meshes and pore size using EM.


    • Tamura-ABD Assays
      • Incubate in albumin containing solution for set time, measure the rate of release of albumin within the hydrogel. Hydrogel with ABD-Tam, and ABD out.
    • CD, IR, before and post vortex of the solution.
    • Flourescence following washes of Tamura.
    • SElective binding to silk sequence regions.
    • Expectation of 100mg per 1L of cell culture.
      • 1ng minimum per well for measureflourescnece.
        • Tamura - 00.5% of total silk, 2%,

0.5% is 25ug per well, 1% 50 ug per well 2% - 100 ug per well.

        • Net STEP: Make hydrogels in PBS tomorrow.