This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Western blot analysis of proteins expressed in yeast
Carmen Bano
Cell Cycle, Universitat València
Corresponding Author
Juan Carlos Igual
Cell Cycle, Universitat València
Corresponding Author
Inma Quilis
Cell Cycle, Universitat València
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Western blot analysis of proteins expression in yeast
Keywords
Labelled protein analysis western blot
- 0.2M NaOH
- 2x SDS-PAGE sample solvent: 4% SDS, 20% glycerol, 0.02% bromophenol blue, 0.1 M DTT, 0.125 M Tris-HCl pH 7.5
- Protein Assay Dye Reagent Concentrate (500-0006 BIO-RAD) for Bradford Assay
- Acrylamide/Bis-acrylamide 30% solution (A3699-100ML, SIGMA-ALDRICH)
- Resolving gel buffer: Tris-HCl 0.75 M pH 8.9, 0.2% SDS, 4 μM EDTA
- Stacking gel buffer: 0.1 M Tris-phosphate pH 6.7, 0.2% SDS, 4 μM EDTA
- Temed (K37762132 741 MERCK)
- PSA: ammonium persulfate 10%
- Electrophoresis buffer: 0.025 M Tris, 0.2 M glycine, 0.1% SDS
- Transfer buffer: 0.025 M Tris, 0.2 M glycine, 0.1% SDS, 20% methanol
- Blocking solution: Skim milk powder (LP0031 OXOID) 2% in TBS-T
- TBS-T solution: 20 mM Tris-HCl pH 7.5, 0.137 M NaCl, 0.01% Tween-20
- Supersignal West Femto Maximum Sensitivity Substrate (34096 Thermo Scientific)
- Incubator
- Corning Tubes Centrifuge
- Eppendorf Tubes Centrifuge
- Thermoblock
- Colorimeter
- Western blot equipment: BIORAD electrophoresis and membrane transfer system
- Stirrer
- Refrigerator and freezer
- Chemiluminescence reader ImageQuant™ LAS 4000mini biomolecular imager (GE Healthcare).
Sample collection
Collect 5x107 cells from the exponentially growing yeast culture
Centrifuge the cells at 2300xg for 2 min. Discard the supernatant
Wash in 1ml of distilled H2O and pass into an eppendorf
- Centrifuge at 2300xg for 2 min. Remove the supernatant with the vacuum. *Freeze the cells at -20°C if the protocol will be continued the next day.
Protein extraction with NaOH
Add 100μL of distilled H2O to resuspend the cell pellet. Then add 100μL of 0.2M NaOH (this treatment disrupts the cell wall) and vortex
Incubate the sample at room temperature for 5-10 min
Centrifuge at 13400xg for 1 min. Remove the supernatant with a vacuum pump
Add 50μL of the 2X SDS-PAGE sample solvent and vortex until to resuspend
Place the samples on the thermoblock at 95°C for 5 min
Centrifuge at 800xg for 10 min at 4°C
- Carefully collect the supernatant (protein extract) – Do not touch the pellet- and pass it into a new eppendorf. Keep these eppendorfs on ice or at -20°C if the protocol will be continued the next day Sample normalization
- Normalization of the samples is performed based on a Bradford assay Each eppendorf should contain:
0.5μL of the protein sample to be quantified
800μL distilled H2O
200μL Protein Assay Dye Reagent
Leave the samples for 5 min at room temperature
Measure absorbance at A595.
Equivalent amount of each protein sample is loaded into PAGE- gel based on this measurement
Electrophoresis
Prepare the SDS-PAGE polyacrylamide gels and place them in the electrophoresis system
Run the electrophoresis at 100V for 2 h approximately depending on the migration you need
Membrane Transfer
Take a tray and pour Transfer Buffer
Place the plastic holder required to make the membrane sandwich
For a wet-BioRad transfer system, place a nitrocellulose membrane in contact with the acrylamide gel between 2 filter papers on top and bottom making sure all have been bathed in transfer buffer. To avoid bubbles being trapped, you may press down with a glass rod. Close the sandwich
Place the sandwich on the correct direction in the sandwich support. Place the whole apparatus into the transfer system. Ensure to place some ice blocks to avoid over-heating. Fill the container with transfer buffer
Set the voltage at 100V and run for 45 min
Protein Detection
Wash the membrane with TBS-T buffer (and place it with agitation)
Incubate with the blocking agent for 1h
Discard the blocking agent and pour the primary antibody mixture onto the membrane. Incubate the membrane with the primary antibody overnight
Prepare the secondary antibody mixture with the corresponding dilution required
Discard the primary antibody mixture. Wash the membranes 3 x 15 min with TBS-T
Discard TBS-T and add the secondary antibody. Incubate the membranes with the secondary antibody for 1h
Discard the secondary antibody and again, wash the membranes 3 x 15 min with TBS-T
Blots are developed using the Supersignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). Bands are observed in a ImageQuant™ LAS 4000mini biomolecular imager (GE Healthcare).
PRIMARY ANTIBODY SECONDARY ANTIBODY
Monoclonal anti-HA peroxidase 3F10 antibody
(Roche Diagnostics, 12013819001)
Diluted 1:5000 Not required
Monoclonal anti-c-myc 9E10 antibody
(Roche Diagnostics, 1667149)
Diluted 1:5000 Goat Anti-mouse IgG (H+L)
Horseradish Peroxidase conjugate
(170-6516, Pierce Antibody, Thermo Scientific) Diluted 1:20000
Monoclonal anti-GFP
(Roche Diagnostics, 11814460001)
Diluted 1:5000 Goat Anti-mouse IgG (H+L)
Horseradish Peroxidase conjugate
(170-6516, Pierce Antibody, Thermo Scientific) Diluted 1:20000
Monoclonal anti-FLAG M2
(Sigma-Aldrich, F3165)
Diluted 1:10000 Goat Anti-mouse IgG (H+L)
Horseradish Peroxidase conjugate
(170-6516, Pierce Antibody, Thermo Scientific) Diluted 1:20000
Monoclonal anti Cdc2 p34 (PSTAIRE)
(Santa Cruz Biotechnology Inc. SC-53)
Diluted 1:2000 Goat Anti-rabbit IgG (H+L)
Horseradish Peroxidase conjugate
(31460, Pierce Antibody, Thermo Scientific)
Diluted 1:20000
Version History
CURRENT STATUS: Posted
Version 1
Posted 29 Jan, 2019
Metrics
Comments: 0
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Method Article
Western blot analysis of proteins expressed in yeast
Carmen Bano
Cell Cycle, Universitat València
Corresponding Author
Juan Carlos Igual
Cell Cycle, Universitat València
Corresponding Author
Inma Quilis
Cell Cycle, Universitat València
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 29 Jan, 2019
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Western blot analysis of proteins expression in yeast
- 0.2M NaOH
- 2x SDS-PAGE sample solvent: 4% SDS, 20% glycerol, 0.02% bromophenol blue, 0.1 M DTT, 0.125 M Tris-HCl pH 7.5
- Protein Assay Dye Reagent Concentrate (500-0006 BIO-RAD) for Bradford Assay
- Acrylamide/Bis-acrylamide 30% solution (A3699-100ML, SIGMA-ALDRICH)
- Resolving gel buffer: Tris-HCl 0.75 M pH 8.9, 0.2% SDS, 4 μM EDTA
- Stacking gel buffer: 0.1 M Tris-phosphate pH 6.7, 0.2% SDS, 4 μM EDTA
- Temed (K37762132 741 MERCK)
- PSA: ammonium persulfate 10%
- Electrophoresis buffer: 0.025 M Tris, 0.2 M glycine, 0.1% SDS
- Transfer buffer: 0.025 M Tris, 0.2 M glycine, 0.1% SDS, 20% methanol
- Blocking solution: Skim milk powder (LP0031 OXOID) 2% in TBS-T
- TBS-T solution: 20 mM Tris-HCl pH 7.5, 0.137 M NaCl, 0.01% Tween-20
- Supersignal West Femto Maximum Sensitivity Substrate (34096 Thermo Scientific)
- Incubator
- Corning Tubes Centrifuge
- Eppendorf Tubes Centrifuge
- Thermoblock
- Colorimeter
- Western blot equipment: BIORAD electrophoresis and membrane transfer system
- Stirrer
- Refrigerator and freezer
- Chemiluminescence reader ImageQuant™ LAS 4000mini biomolecular imager (GE Healthcare).
Sample collection
Collect 5x107 cells from the exponentially growing yeast culture
Centrifuge the cells at 2300xg for 2 min. Discard the supernatant
Wash in 1ml of distilled H2O and pass into an eppendorf
- Centrifuge at 2300xg for 2 min. Remove the supernatant with the vacuum. *Freeze the cells at -20°C if the protocol will be continued the next day.
Protein extraction with NaOH
Add 100μL of distilled H2O to resuspend the cell pellet. Then add 100μL of 0.2M NaOH (this treatment disrupts the cell wall) and vortex
Incubate the sample at room temperature for 5-10 min
Centrifuge at 13400xg for 1 min. Remove the supernatant with a vacuum pump
Add 50μL of the 2X SDS-PAGE sample solvent and vortex until to resuspend
Place the samples on the thermoblock at 95°C for 5 min
Centrifuge at 800xg for 10 min at 4°C
- Carefully collect the supernatant (protein extract) – Do not touch the pellet- and pass it into a new eppendorf. Keep these eppendorfs on ice or at -20°C if the protocol will be continued the next day Sample normalization
- Normalization of the samples is performed based on a Bradford assay Each eppendorf should contain:
0.5μL of the protein sample to be quantified
800μL distilled H2O
200μL Protein Assay Dye Reagent
Leave the samples for 5 min at room temperature
Measure absorbance at A595.
Equivalent amount of each protein sample is loaded into PAGE- gel based on this measurement
Electrophoresis
Prepare the SDS-PAGE polyacrylamide gels and place them in the electrophoresis system
Run the electrophoresis at 100V for 2 h approximately depending on the migration you need
Membrane Transfer
Take a tray and pour Transfer Buffer
Place the plastic holder required to make the membrane sandwich
For a wet-BioRad transfer system, place a nitrocellulose membrane in contact with the acrylamide gel between 2 filter papers on top and bottom making sure all have been bathed in transfer buffer. To avoid bubbles being trapped, you may press down with a glass rod. Close the sandwich
Place the sandwich on the correct direction in the sandwich support. Place the whole apparatus into the transfer system. Ensure to place some ice blocks to avoid over-heating. Fill the container with transfer buffer
Set the voltage at 100V and run for 45 min
Protein Detection
Wash the membrane with TBS-T buffer (and place it with agitation)
Incubate with the blocking agent for 1h
Discard the blocking agent and pour the primary antibody mixture onto the membrane. Incubate the membrane with the primary antibody overnight
Prepare the secondary antibody mixture with the corresponding dilution required
Discard the primary antibody mixture. Wash the membranes 3 x 15 min with TBS-T
Discard TBS-T and add the secondary antibody. Incubate the membranes with the secondary antibody for 1h
Discard the secondary antibody and again, wash the membranes 3 x 15 min with TBS-T
Blots are developed using the Supersignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). Bands are observed in a ImageQuant™ LAS 4000mini biomolecular imager (GE Healthcare).
PRIMARY ANTIBODY SECONDARY ANTIBODY
Monoclonal anti-HA peroxidase 3F10 antibody
(Roche Diagnostics, 12013819001)
Diluted 1:5000 Not required
Monoclonal anti-c-myc 9E10 antibody
(Roche Diagnostics, 1667149)
Diluted 1:5000 Goat Anti-mouse IgG (H+L)
Horseradish Peroxidase conjugate
(170-6516, Pierce Antibody, Thermo Scientific) Diluted 1:20000
Monoclonal anti-GFP
(Roche Diagnostics, 11814460001)
Diluted 1:5000 Goat Anti-mouse IgG (H+L)
Horseradish Peroxidase conjugate
(170-6516, Pierce Antibody, Thermo Scientific) Diluted 1:20000
Monoclonal anti-FLAG M2
(Sigma-Aldrich, F3165)
Diluted 1:10000 Goat Anti-mouse IgG (H+L)
Horseradish Peroxidase conjugate
(170-6516, Pierce Antibody, Thermo Scientific) Diluted 1:20000
Monoclonal anti Cdc2 p34 (PSTAIRE)
(Santa Cruz Biotechnology Inc. SC-53)
Diluted 1:2000 Goat Anti-rabbit IgG (H+L)
Horseradish Peroxidase conjugate
(31460, Pierce Antibody, Thermo Scientific)
Diluted 1:20000