20.109:TAP-tags

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Revision as of 08:59, 25 June 2008 by Nkuldell (talk | contribs) (New page: ==New Protein Engineering Module for F08== Point of departure is to ask if tags are actually neutral wrt protein function ===Rough sketch of labs=== *Part 1: TAP tag **non-essential SAGA ...)
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New Protein Engineering Module for F08

Point of departure is to ask if tags are actually neutral wrt protein function

Rough sketch of labs

  • Part 1: TAP tag
    • non-essential SAGA subunits (essential likely in many complexes so purif profile complex + tag has many consequences)
    • unknown ORFs changed during microarray of SAGA del or mtRNT1

1-1 Design primers
1-2 PCR
1-3 Transform
1-4 Confirm integration by colony PCR

    • Alternatively, could just give students tagged version.
  • Part 2: Western
    • need + TAP control
    • use Sigma Ab to protA (PAP P-2026, anti-ProtA)
    • 1.5 ml ON culture, vortex 3X30' with glass beads and add 100 ul LB from Methods (2001) 24:218
  • Part 3: Phenotypes
    • compare parental, K/O, TAP-tagged
    • examine GAL, Spt (his4-917d), TS, CS
  • Part 4: Array
    • compare parental to TAP-tagged

Could also include

Mass Spec analysis description/tour/lab?
Tour of protein purif facilty

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