20.109:TAP-tags: Difference between revisions

Revision as of 13:01, 25 June 2008

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

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 facility

FB2305
- MC1066+pBS1539 (AmpR+URA3-Kl, TAP tag)
FB2306
- MC1066+pBS1479(AmpR+TRP1-Kl, TAP tag)
- ref for seq of TRP1 from Kluyveromyces lactis here

FY631 = Parental for TAP tagging
- MATa, his4-917d, lys2-173R2, leu2d1, ura3-52, trp1d63
FY2714
- MATa, HA-SPT3-TAP::TRP1, his4-917d, lys2-173R2, ura3D0 or -52, leu2D1 or D0, trp1D63
FY2031
- MATa, HA-SPT7-TAP::TRP1, his4-917d, lys2-173R2, ura3D0, leu2D1, trp1D63
FY2305
- MATa, SPT10-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
- not his4-912d
FY2306
- MATa, SPT21-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
- not his4-912d

General format for

@@ Line 29: / Line 29: @@
 *FB2306
 **MC1066+pBS1479(AmpR+TRP1-Kl, TAP tag)
+** ref for seq of TRP1 from Kluyveromyces lactis [http://www.ncbi.nlm.nih.gov/pubmed/2538971 here]
 [[Image:TAPplasmids.png|thumb|left|600px| from Methods 2001 article[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WN5-456JRHJ-17&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=53bcb4f821b860dce449e6105fb83081]]]
 ====Yeast====