Application
Western blot
Sample
Saccharomyces cerevisiae Cell lysate - whole cell (yeast cell lysate)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
10 µg
Specification
yeast cell lysate
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
Other product details
Incubation time
2 hour(s) and 0 minute(s) · Temperature: 22°C · Diluent: 5% Milk
Dilution
1/5000
Secondary antibody
Secondary antibody
None used
Detection
Detection method
ECL
Exposure
1 minute(s) and 0 second(s)
Bands
Specific: 65 kDa
Additional data
Additional Notes
In the experiment the protein of interest is tagged with a single FLAG tag and expressed with a constitutive promoter. Sample 1, 2, 3, 4 are the lysates from the strains expressing the tagged protein. The experiment was done with cells grown to 1.5 OD units and lysed using glass beads with the lysis buffer containing MOPS, SDS, Urea and EDTA. 0.4 OD equivalent of cells were analyzed on the SDS-PAGE and western blotting was preformed with the Abcam anti-Flag antibody which is tagged with HRP. Nitrocellulose membrane was used for the blotting.
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投稿 Mar 26 2014