Biology Reference
In-Depth Information
with the bait construct and a prey empty vector is advisable)
dissolved in deionized sterile water (300-600 ng of each plas-
mid in a fi nal volume of 75
fl
2. Mix vigorously and incubate at 30 °C for 20-30 min.
3. Mix by gently inverting the tubes and transfer to a preheated
42 °C circulating water bath or thermo block. Incubate for
30 min.
4. Allow the cells to cool at room temperature for 1-2 min.
5. Centrifuge the cells at 3,000 × g for 3 min at room tempera-
ture. Discard the supernatant and resuspend the pellet in
900
μ
l of deionized sterile water.
6. Spread different amounts of the resuspended cells onto a plate
(10 cm of diameter) containing the appropriate selection
medium to obtain isolated colonies (normally 10 and 100
μ
fl
For pGBKT7, use MM without tryptophan. For pGADT7,
use MM without leucine. For cotransformations use CM.
7. Incubate the plates at 30 °C for 3-5 days.
μ
For detection of the fusion protein, a Western blot of protein
extracts obtained from the transformed yeast could be performed
using Gal4 DNA-BD Monoclonal Antibody (Clontech) or -Myc
Monoclonal Antibody (Clontech).
Detecting Bait Expression
and Toxicity
1. Yeast protein extract can be prepared by growing yeast in MM
without tryptophan, to maintain selection for the bait plasmid,
to OD 600 = 0.8-1.0.
2. Centrifuge 30-40 ml of the culture at 4,000 × g for 3 min to
pellet the cells. Wash the pellet once with cold water, transfer
to an ice-cold screw-cap tube containing 0.5 ml of glass beads
(425-600
μ
m) and resuspend in 200
μ
l of RIPA buffer con-
l to 5 ml of buffer) and
1 mM phenylmethylsulfonyl fl uoride (PMSF). Grind in
FastPrepTM FP120 (BIO 101) at power setting 5.5 for two
15 s intervals separated by 1 min intervals on ice.
3. Add 400
taining protease inhibitor cocktail (50
μ
l RIPA buffer with protease inhibitors and vigor-
ously vortex. Keep on ice. Heat needle in a fl ame and quickly
pierce bottom of the screw-cap tube containing lysate. Make
2-3 holes in each tube and place this 1 min in cooled centri-
fuge (do six tubes at a time). Keep on ice.
4. Centrifuge at 4,000 × g , 2 min in a cooled centrifuge to pellet
the cells. Carefully recover the supernatant containing the pro-
tein extract. Protein concentration can be determined using a
standard Bradford assays.
5. Load 100
μ
g of the protein extract on a polyacrylamide/SDS
gel. The proteins can then be transferred to a fi lter and blotted
with standard immunoblotting (Western) methods [ 12 ].
μ
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