251
The target protein can be detected by immunoblotting using antibodies directed against the tester protein tagRFP. For immunoblotting, crude cell extracts can be prepared by alkaline lysis [27].
1. 1 ml of logarithmically growing cells (A 600 = 1) is treated with
150 μl of alkaline lysis buffer and kept for 10 min on ice.
2. Protein precipitation is induced by addition of 150 μl 55%
(w/v) trichloroacetic acid (TCA) followed by 10 min incubation on ice.
3. The samples are subjected to centrifugation (10 min, 16,000 ×
g) and the supernatant is removed. The pellet is dissolved in 60
μl of high urea buffer by mixing the sample vigorously at 65 °C.
4. The extracts are cleared from cell debris by centrifugation (10
min, 16,000 × g), 10–20 μl of sample is loaded onto an SDSPAGE gel. Standard procedures can be used for SDS-PAGE
and blotting [28, 29].
Cycloheximide is a translational elongation inhibitor that can be
used to stop protein biosynthesis in eukaryotic cells. Thus, the stability of a protein can be inferred from the decay rate after addition
of cycloheximide to logarithmically growing yeast cells. Target
protein levels can be detected by an appropriate assay, e.g.,
immunoblotting.
1. Grow yeast cells in darkness until logarithmic growth phase is
reached.
2. Take first sample (t = 0 h) and subject to alkaline lysis.
3. Add cycloheximide to a final concentration of 200 μg/ml to
the yeast culture to stop protein synthesis.
4. Expose cells to specific illumination conditions, e.g., darkness or
a distinct amount of blue light for the rest of the experiment.
5. Collect equal amounts of sample at several time points that
match the protein decay rate (e.g., every 30 min) and subject
to alkaline lysis.
6. Perform immunoblotting to measure protein abundance over
time.
The target protein RFP can directly detected by fluorescence measurements with a fluorimeter. For the interpretation of the results,
the maturation time of the used fluorescent protein has to be taken
into account.
1. 1 ml of logarithmically growing cells (A 600 = 1) is treated with
100 μl of sodium azide buffer.
2. Samples are subjected to centrifugation (3 min, 500 × g) and
1 ml of the supernatant is removed. The pellet is dissolved in
the remaining supernatant.
3.7 Detection of the
Target Protein RFP
by Immunoblotting
3.8 Measurement
of Protein Degradation
by Cycloheximide
Chase
3.9 Quantification
of Target Protein
Levels
by Fluorescence
Controling Protein Stability with Light
The target protein can be detected by immunoblotting using antibodies directed against the tester protein tagRFP. For immunoblotting, crude cell extracts can be prepared by alkaline lysis [27].
1. 1 ml of logarithmically growing cells (A 600 = 1) is treated with
150 μl of alkaline lysis buffer and kept for 10 min on ice.
2. Protein precipitation is induced by addition of 150 μl 55%
(w/v) trichloroacetic acid (TCA) followed by 10 min incubation on ice.
3. The samples are subjected to centrifugation (10 min, 16,000 ×
g) and the supernatant is removed. The pellet is dissolved in 60
μl of high urea buffer by mixing the sample vigorously at 65 °C.
4. The extracts are cleared from cell debris by centrifugation (10
min, 16,000 × g), 10–20 μl of sample is loaded onto an SDSPAGE gel. Standard procedures can be used for SDS-PAGE
and blotting [28, 29].
Cycloheximide is a translational elongation inhibitor that can be
used to stop protein biosynthesis in eukaryotic cells. Thus, the stability of a protein can be inferred from the decay rate after addition
of cycloheximide to logarithmically growing yeast cells. Target
protein levels can be detected by an appropriate assay, e.g.,
immunoblotting.
1. Grow yeast cells in darkness until logarithmic growth phase is
reached.
2. Take first sample (t = 0 h) and subject to alkaline lysis.
3. Add cycloheximide to a final concentration of 200 μg/ml to
the yeast culture to stop protein synthesis.
4. Expose cells to specific illumination conditions, e.g., darkness or
a distinct amount of blue light for the rest of the experiment.
5. Collect equal amounts of sample at several time points that
match the protein decay rate (e.g., every 30 min) and subject
to alkaline lysis.
6. Perform immunoblotting to measure protein abundance over
time.
The target protein RFP can directly detected by fluorescence measurements with a fluorimeter. For the interpretation of the results,
the maturation time of the used fluorescent protein has to be taken
into account.
1. 1 ml of logarithmically growing cells (A 600 = 1) is treated with
100 μl of sodium azide buffer.
2. Samples are subjected to centrifugation (3 min, 500 × g) and
1 ml of the supernatant is removed. The pellet is dissolved in
the remaining supernatant.
3.7 Detection of the
Target Protein RFP
by Immunoblotting
3.8 Measurement
of Protein Degradation
by Cycloheximide
Chase
3.9 Quantification
of Target Protein
Levels
by Fluorescence
Controling Protein Stability with Light