Tuesday, November 18, 2003

Description
nup4a-313 Shuttling in Yeast


Procedure
Method
1. Transform a plasmid containing YFG-GFP under the control of the galactose inducible promoter (pGAL1-10)a into a nup49-313 strainb
2. Grow the transformants in 2% glucose containing dropout media to saturation at 25C
3. Dilute (1:200 dilution) and grow the cells in 2% raffinose containing dropout media at 25C.a
4. When the cells reach 1 x 107 cells/ml, add 2% galactose to the cultures and incubate at 25C for 3 hours.a
5. Wash the cells once with dH2O and resuspend the cells in dropout media containing 2% glucose.a
6. Incubate the cells at 25C for 2 hours.a
7. Split each culture and incubate one half of the culture at 25C and the other half of the culture at 37C for 5 hours.
8. Remove 1 ml of cells from each culture, wash once with dH2O, and resuspend in 25-100 ml of dH2O
9. Place 3 ml of washed cells onto a glass slide and analyze nuclear export by fluorescence microscopy.


aAlternatively, cells expressing YFG-GFP under the control of its endogenous promoter (pYFG) can be used in the assay. Steps 3-6 should be modified to include shifting the cells for 2-3 hours until they are in mid log phase followed by incubation of the cells with 100 mM cyclohexamide before splitting and shifting the cells.
bThis assay can be coupled with other mutants to examine whether other proteins affect nuclear export of YFG-GFP



Recipes
PEG/TE/LiOAc
TE/LiOAc
DMSO
2% glucose
2% galactose
Dropout media
2% raffinose
Deionized water
25 x 75 x 1 mm frosted microscope slides
24 x 40 mm micro cover slips
Phase contrast and fluorescence microscope


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