TITLE: Western Blot
AUTHORS: Karyna Tarasova, Angkana Kidtiwong, Sinan Gültekin, Florien Jenner

[DESCRIPTION]
Western Blot - protein expression assessment

[BEFORE_START]
Before start please check the antibody datasheet provided by the manufacturer. 

[STEPS]
SECTION: Sample preparation
1. Protein Quantification - BCA, Quibit measurment
SECTION: Sample preparation
2.        After determining how much protein to load, add an equal volume of 2X Laemmli sample buffer, in order to reduce and denature the samples, unless the online antibody datasheet indicates that non-reducing and non-denaturing conditions should be used.
SECTION: Sample preparation
2.1.        Do a spindown for 10 s. 
SECTION: Loading and running the gel eletrophoresis
4. Gel loading and eletrophoresis
SECTION: Loading and running the gel eletrophoresis
4.1.        Assemble the electrophoresis:
SECTION: Loading and running the gel eletrophoresis
4.2. a.       Remove the comb from the gels 
SECTION: Loading and running the gel eletrophoresis
4.3. b.       Fill the inner and outer buffer chambers with running buffer. Fill the inner buffer chamber of each core with 200 mL of Running buffer. 
SECTION: Loading and running the gel eletrophoresis
4.4.        Load equal amounts of protein into the wells of the SDS-PAGE gel, along with molecular weight marker (5-10µL). 
SECTION: Loading and running the gel eletrophoresis
4.5.        Connect the electrophoresis cell to the power supply and perform electrophoresis.
SECTION: Loading and running the gel eletrophoresis
4.6.        Run the gel at 150V for 1h 15min, or until the blue reaches the end of the gel. 
SECTION: Loading and running the gel eletrophoresis
4.7.        After electrophoresis is complete, turn the power supply off and disconnect the electrical leads. Pop open the gel cassettes and remove the gel by floating it off the plate into Transfer buffer. 
SECTION: Transfer
5. Prepare the transfer buffer according to the manufacturer’s instructions.
SECTION: Transfer
6. The membrane can be either nitrocellulose or PVDF. Activate PVDF with methanol for 30sec and wash heavily with water.  Rinse with transfer buffer before preparing the stack.
SECTION: Transfer
7. Prepare the sandwich according to the manufacturer’s instructions. Make sure you wet the BioRad white paper filter in transfer buffer before you prepare the sandwich.  IMPORTANT: The gel should be on the black side of the sandwich and the membranes should be on the red side. Be sure you remove the air bubbles with a BioRad roller.
SECTION: Transfer
8. Start the run at 370 Å for 1h30min in an ice cold bath (put a magnet inside the cassette before starting the transfer and surround the cassette with ice after closing).
SECTION: Antibody staining
9. Transfer of proteins to the membrane can be checked using Ponceau S staining before the blocking step. If necessary, use Ponceau S during 30 min (under slow agitation). IMPORTANT: After 30 min wash the membrane with H2O three times, 5 min each (under mid-fast agitation). Take a print of the membrane. If necessary, prepare a solution of 0.1M NaOH in H2O in order to remove the remaining Ponceau. Wash the membrane with H2O to remove the NaOH solution.
SECTION: Antibody staining
10. Block the membrane for 1 h at room temperature using blocking buffer, Milk 5% (under slow agitation).
SECTION: Antibody staining
11. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer, Milk 5%. The recommend condition is overnight incubation at 4°C (under slow agitation); other conditions can be optimized.
SECTION: Antibody staining
12. Wash the membrane three times with TBST, 10 min each (under mid-fast agitation).
SECTION: Antibody staining
13. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer, Milk 5%, at room temperature for 1 h (under slow agitation).
SECTION: Antibody staining
14. Wash the membrane three times with TBST, 10 min each (under mid-fast agitation).
SECTION: Antibody staining
15. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.
SECTION: Antibody staining
16. Acquire image using darkroom development techniques for chemiluminescence.
SECTION: Sample preparation
3.       Boil each cell lysate in sample buffer at 70°C for 10 min. Place the samples at room temperature and wait for a few seconds.