Comparative Physiology @ Guelph
The objective for doing the Western Blot is to identify and compare the proteins separated from the fraction collection.
- Plastic bag and sealer
- Belly dancer
- Thick transfer paper
- Small containers for soaking papers and gels
- Scissors cleaned with 70% ethanol
- Tweezers cleaned with 70% ethanol
- Nitrocellulose membrane
- Transfer unit with power supply
- Fraction collection tube for rolling out bubbles
- 1000P Micropipette with tips
- Paper towel and Kimwipes
- Electrophoresis cassette
- 1x transfer buffer (from 10x transfer buffer: 25mM Tris, 192 mM glycine)
- Blocking buffer (5% skim milk, TBST)
- Primary antibody
- Secondary antibody
- ECL mixture components
- Make a duplicate of the gel when doing the SDS Page. Run them in the same conditions. For good band resolution with these concentrations of antibodies, a good starting point is to load approximately 4ul of the fraction with 4ul of 6M urea and 4ul of 3x SDS. This concentration will depend on the concentration of the fraction which can be estimated according to the peak height of the column run, and may be adjusted. A good starting point is about 0.25 mg/mol. The amounts noted here are for a half gel size. Adjust amounts of antibody dilution and time for transfer accordingly for a full gel.
- Prepare three small dishes containing the transfer buffer, methanol and ddH2O.
- Wear gloves so as to not damage the membrane. Rinse transfer unit with ddH2O.
- Carefully remove the gel from the mold, and cut to size. Cut off the top left corner to determine orientation.Rinse with ddH2O. Use gel to measure out and cut 6 thick pieces of transfer paper, as well as one piece of nitrocellulose paper (membrane) per gel.
- Soak 3 filter papers in 1x transfer buffer. Soak lower gel in 1x transfer buffer for ~10 minutes on the belly dancer. Soak membrane curved side down in methanol (place methanol in coomassie waste when finished), shake for 1 min. Rinse membrane with ddH2O until beading is gone.
- Place the gel on top of 3 filter papers, being sure to avoid making any bubbles, and repeat top left corner cut to maintain orientation. Place on transfer unit.
- Soak the remaining transfer papers and the membrane in the transfer buffer.
- Place the membrane on top of the gel, curved side against the gel, without introducing bubbles. Flip the stack over, and place on bottom filter papers in the centre of the transfer unit. Trim the sandwich to be the same size as the gel. Pour some transfer buffer on the sandwich and roll out any bubbles with a tube.
- Attach the transfer unit with the Thermo EC and set the time to approximately 30 minutes for a thin half gel, the maximum current to 500mA, and the maximum voltage to 30 V. Close the lid, and wait for the transfer.
- While the transfer is happening, make 5% blocking buffer using skim milk powder in ~ 10 mL TBST. Vortex in a falcon tube to solublize and store in the fridge until needed. Cut a plastic bag to size that will easily fit the membrane.
- One the transfer is complete, turn off the voltage, and check that the ladder has transferred. Remove the membrane with tweezers, mark the ladder, and place in plastic bag. Bring the blocking buffer and bag containing membrane to the sealer in room 3406. Seal 3 sides, pour in the blocking buffer and then seal up the final side with as little air in the bag as possible. Place the labelled bag on the rocker in the cold room overnight, with the transferred side up.
- The next day, thaw the antibodies needed. Prepare the primary antibody solution at the desired concentration in blocking buffer. Lay down clean parafilm flat on a glass slide, and put 750ul of solution into a puddle on the slide with no bubbles. Using tweezers, place the membrane transferred side down on the antibody solution. Cover with a Tupperware container, and allow to incubate at room temp for 3 hours.
- In a small container, rinse in 3% blocking buffer, then TBST twice for 10 minutes each on the shaker.
- Repeat the soaking in 750ul of the secondary antibody at a concentration of 1:10000. Allow to incubate 1h covered, at room temperature. Rinse 3x in TBST for 10 minutes each.
- Prepare cassette by wiping down with damp Kimwipe. Mix ECL mixture 1:1 in falcon tube, with 2 ml final mixture per half gel size. Blot the membrane on a paper towel, and place on tinfoil or parafilm surface phase side up. Cover with 2 ml of the mixture for 1 min. Blot membrane with paper towel, and place between plastic sheets in the cassette. Ensure there are no bubbles.
- Bring a key, the box of film, cassette, a marker, scissors, and timer to room 2402C. Knock on the door before entering. Take 3 buckets (labeled Fixer, Water, developer) and two containers (labeled Fixer and Developer) underneath the sink, and place them on the bench. Turn lights off, turn the safe light on, and then pour the fixer and developer (500 mL approximately) in their respective buckets. Pour water (500 mL approximately) in the water bucket. Prepare half a sheet of film with cut corner, and line it up in the cassette for about 10 seconds.Develop the film by placing it (using tweezers, wearing gloves) in the developer for 10 sec (agitation). Rinse in water for 10 sec (agitation) and then place in the fixer for 10 sec (agitation). The lights can then be turned on then. Hang the film in the dryer cabinet and turn it on (5 min).