TITLE: Protein Extraction and western blotting
AUTHORS: t.a.mcdougall 

[STEPS]
?. [Protein Extraction]
Whole cell protein was extracted from cells by treating cell pellets with Radio Immunoprecipitation Assay buffer (RIPA buffer: 1M NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1%SDS, 50mM pH 7.4 Tris in ddH2O) was prepared according to the Cold Spring Harbor and supplemented with 1µl of Protease Inhibitor Cocktail from Roche per 100µl of RIPA buffer. RIPA buffer was added at a concentration of 500µl per ~2x106 cells.
?. [Protein Extraction]
Samples were gently agitated by shaking for 30 minutes at 4℃ to allow for the breakdown of cells, then were centrifuged at max speed (>14,000 rcf) at 4℃ for 20 minutes.
?. [Protein Extraction]
The supernatant containing the protein component was transferred to a fresh microcentrifuge tube to be loaded immediately into an SDS-PAGE gel or stored at -20℃.
?. [SDS-PAGE gel]
In preparation for running protein samples on the SDS-PAGE gel a 1.5mm glass spacer plate and cover plate were sandwiched together in a casting clamp and placed upon a gel casting stand (all from Bio Rad).
?. [SDS-PAGE gel]
The volume within the plates was filled with water and allowed to sit for several minutes to ensure that there was no leakage. Once it was confirmed that there were no leaks the water was poured out.
?. [SDS-PAGE gel]
10ml of 12% acrylamide lower running gel was prepared consisting of 4.2ml of 29:1 acrylamide/bis-acylamide (Bio Rad), 2.5ml of 4x lower gel buffer (1.5M Tris and 0.4%SDS in distilled water, pH of 8) and 3.3ml of distilled water. This was mixed thoroughly then 40µl of 10% ammonium persulfate (prepared freshly each time) as well as 10µl of TEMED from Fisher Scientific was added.
?. [SDS-PAGE gel]
The lower running gel solution was immediately poured into the gel casting plates to within 1cm of the top. The final cm of the plates was filled with 100% isopropanol. The lower gel solidified for a minimum of 30 minutes.
?. [SDS-PAGE gel]
3ml of upper gel mixture consisting of 0.75ml 29:1 acrylamide/bis-acylamide, 0.45ml of 4x upper gel buffer (0.5M Tris and 0.4%SDS in distilled water, pH of 6.8), 1.8ml of distilled water, 10µl of of 10% ammonium persulfate and 5µl of TEMED(Fisher Scientific) was prepared once the lower gel had solidified.
?. [SDS-PAGE gel]
The isopropanol covering the lower gel was poured off completely, exposing the top of the lower gel. The upper gel mixture was poured on top, filling the remaining centimeter in the gel plates. A 1.5mm gel comb that accompanied the spacer plate was then inserted into the top of the plates. This upper gel mixture solidified over the course of 30 minutes.
?. [SDS-PAGE gel]
The solid gel and its glass case were removed from the casting stand and clamp and were fitted into the electrode assembly (Bio Rad) with the short plates facing inwards; the comb was then removed. If only one gel was being run then a mock spacer plate and short plate combination was inserted on the other side of the electrode assembly with short plate on the inside. This created a watertight container that was fitted into the Bio Rad buffer tank and filled to overflowing with Running buffer.
?. [SDS-PAGE gel]
Running buffer was produced 1 liter at a time by diluting 100ml of 10x Running buffer (0.25M Tris, 1.92M Glycine and 1% w/v SDS in distilled water) in 900ml distilled water. The tank itself was then filled with the remaining Running buffer.
?. [SDS-PAGE gel]
Once the gel was prepared for electrophoresis, 15µl of each protein extract was mixed with 2X SDS gel-loading buffer (4% SDS, 0.2% bromophenol blue, 20% glycerol and 200mM dithiothreitol) according to the Cold Spring Harbor protocol and the resulting mixture was heated to 95℃ for 2-3 minutes
?. [SDS-PAGE gel]
The protein mixture was loaded into each well of the gel, and 5µl BenchMark Pre-stained Protein Ladder from Thermo Fisher was loaded into one of the wells abutting the samples.
?. [SDS-PAGE gel]
The gel was run at 180 volts until the bromophenol blue dye in the various samples had nearly reached the bottom of the gel but had not run off. The bottom band of the Pre-stained Protein Ladder corresponded to ~10kDa and so it was ensured that there was always a significant gap between this band of the ladder and the bottom of the gel to ensure that the histones had not been run off the gel.
?. [Western blotting]
Transfer buffer was prepared by combining 100ml of 10x Transfer buffer (0.25M Tris and 1.9M glycine in distilled water and stored at 4°C), 200ml of methanol and 700ml of distilled water.
?. [Western blotting]
The gel was removed from the tank, electrode assembly and spacer plates. The gel was immersed for 5 minutes in cold Transfer buffer and the transfer system was prepared.
?. [Western blotting]
The Bio Rad transfer holder cassette was arranged with the black frame (which would face the negative electrode) on one side and the clear frame (which would face the positive electrode) on the other. A foam pad from Bio Rad was placed on top of the black frame and upon that was placed a piece of the filter paper supplied in the Nitrocellulose/Filter Paper Sandwich from Thermo Fisher.
?. [Western blotting]
The now soaked gel was placed carefully upon that piece of filter paper so as to be completely supported by it and not touching any part of the sponge beneath.
?. [Western blotting]
Carefully and using metal tweezers, the 0.2µm nitrocellulose was first briefly soaked in Transfer buffer then placed on top of the gel. Pressure was applied with a smooth flat implement to remove any gaps that might exist between the nitrocellulose and gel and a small amount of transfer buffer was poured on top to ensure the nitrocellulose was completely soaked.
?. [Western blotting]
The other piece of filter paper was placed on top of the nitrocellulose to cover it completely. An additional piece of spongy foam was then placed on top of the filter paper. The clear frame was then folded over and fastened to the black frame and the cassette was fitted into the Bio Rad Core Assembly Module for protein transfer with the black of the cassette facing the black side of the module.
?. [Western blotting]
The Core Assembly Module was inserted into the Bio Rad protein transfer tank and a cold ice pack was placed in the transfer tank as well. The tank was then filled with Transfer buffer and was hooked up to a Bio Rad PowerPac HC power supply.
?. [Western blotting]
Protein transfer was carried out at 90 volts for one hour at 4℃ with constant gentle agitation of the Transfer buffer using a magnetic stir bar to ensure dispersion of heat.
?. [Western blotting]
After the one hour the protein bound nitrocellulose membrane was transferred into a container filled with 40ml of blocking buffer consisting of Tris buffered saline (TBS) with 0.1% v/v Tween-20 and 3% Bovine Serum Albumin (BSA: 1.2 grams in 40ml TBS).
?. [Western blotting]
The membrane was gently rocked back and forth for one hour in the blocking buffer to prevent the non-specific absorption of antibodies into the nitrocellulose.
?. [Western blotting]
After blocking, the nitrocellulose was placed inside a 50ml falcon tube containing 20ml of primary antibody solution (3% w/v BSA, 0.1% v/v Tween-20 in TBS plus the relevant antibodies). The primary antibodies used in the cited study are shown here.   Primary western
  blotting antibodies    Target    Company    Catalog #    Lot #    Host species    Dilution    H3K27me3     Diagenode    C15410069    A1824D    rabbit    1:500    H2AK119ub    Sigma-Aldrich    05-678    2591849    mouse    1:2000    H4    Upstate    25296    07-108    rabbit    1:2000    β-actin    Invitrogen    MA5-15739    TC263471    mouse    1:5000
Primary western
  blotting antibodies    Target    Company    Catalog #    Lot #    Host species    Dilution    H3K27me3     Diagenode    C15410069    A1824D    rabbit    1:500    H2AK119ub    Sigma-Aldrich    05-678    2591849    mouse    1:2000    H4    Upstate    25296    07-108    rabbit    1:2000    β-actin    Invitrogen    MA5-15739    TC263471    mouse    1:5000
?. [Western blotting]
The membrane was incubated in this solution overnight at 4℃ on a tube rotator to allow even coating of the entire surface of the membrane with antibodies.
?. [Western blotting]
The next day the membrane was retrieved from its falcon tube and was washed four times for 5 minutes each in room temperature TBST (0.1% v/v Tween-20 in TBS). During each wash the nitrocellulose membrane was placed on a rocker to help wash off any unbound proteins.
?. [Western blotting]
The nitrocellulose membrane was then placed in a container containing 20ml of secondary antibody solution (3% w/v BSA, 0.1% v/v Tween-20 in TBS plus the secondary antibody of interest. The membrane was incubated in the secondary antibody solution for one hour at room temperature on a gently rocking rocker.   Secondary fluorescent western blotting antibodies    Target    Company    Catalog #    Lot #    Host species    Dilution    Rabbit
  antibodies    LI-COR    925-68071    B81113-01    goat    1:10,000    Mouse
  antibodies    LI-COR    925-32210    926-32201    goat    1:10,000
Secondary fluorescent western blotting antibodies    Target    Company    Catalog #    Lot #    Host species    Dilution    Rabbit
  antibodies    LI-COR    925-68071    B81113-01    goat    1:10,000    Mouse
  antibodies    LI-COR    925-32210    926-32201    goat    1:10,000
?. [Western blotting]
Following this secondary incubation, the membrane was washed twice with TBST then once in TBS for 5 minutes each at room temperature.
?. [Western blotting]
The protein and antibody bound membrane was then imaged at the relevant wavelengths using an LI-COR Odyssey machine from BioAgilytix and the software package, Image Studio.