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Western Blotting
Gel casting preparation
1. Prepare the caster: 2. Prepare the clamps 3. Construct the gel sandwich: two perfectly clean unchipped glass plates and two spacers (75% ethanol wash and dry). 4. Loose the screw of the clamps. 5. Secure the sandwich with clamps. 6. Inspect the bottom of the sandwich to make sure that edges are aligned flush in order to ensure a complete seal. 7. Place the laminated gasket into the casting cradle with the foam side down.. Place the glass plate assembly in the casting cradle, screw side facing out. 8. Use a cam to fix it.

Acrylamide gel preparation

Resolving gel:
1. Prepare the monomer solution and pour the gel. (pay attention to the pipet: plastic or glass). 2. Pipet the solution into one corner of the sandwich, taking care not to introduce any air bubbles. 3. Overlay the resolving gel with a thin layer of Isopropanol (500 l) to prevent exposure of the top surface of the gel solution to atmospheric oxygen. 4. Allow the gel to polymerize for a minimum one hour.

Stacking gel:
1. Remove the overlay by rinsing the top of the gel several times with distilled water. Dry it by sucking. Remove residual liquid by blotting one corner with a lint-free tissue. 2. Put the comb on the top side of the sandwich. 3. Pour the stacking gel onto the resolving gel with a disposable pipet to a level about 2 mm from the top of the glass plate.

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4. Introduce a comb into the sandwich and push it. Take care not to trap air under the teeth. Allow a minimum of one hour for the gel to polymerize.

Sample preparation
Factors which affect the amount of sample: the thickness of the gel, the sensitivity of the detection method used, the amount of sample expected in each band. 1. Prepare the well: Remove the comb gently rocking side to side and then lifting it out. Carefully rinse each well with running buffer . Fill each well with electrophoresis buffer. 2. Prepare the sample. (2X Sample buffer : 10% 2-Mercaptoethanol = 1:1, final go 1X and 5%) (Marker: 1l + 49l sample buffer) 3. Boil the sample for 100 C, ~5 min. Remember to lock the lid of the sample tube. 4. Mix the sample and spin it down.

Final assembly
1. Rinse both the buffer chambers with water and distilled water thoroughly before each use. 2. Add running buffer 200 ml. Install the gel sandwich in the lower buffer chamber. 3. Carefully fill each sample well with electrophoresis buffer. Then underlay prepared sample into the wells using a fine-tipped microsyringe or pipet tip. 4. Add running buffer one drop to one well till the wells are filled out. 5. Attach the upper buffer chamber and press the slotted gasket into the grooves for a precise fit. 6. Pour 10ml running buffer into the upper chamber groove, taking care not to disturbing the sample. Then Fill 300 ml the buffer into it. 7. Safety lid installation. Take care the anode-red (+), and the cathode-black (-).

Resolving the sample

1. Current: 5mA or higher. 2. Voltage: 40-60mV.

Yu Lab. 3. Time: overnight. 4. Room: cold room. 5. Record each run.

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After electrophoresis
1. Turn off the power once the tracking dye reaches the bottom of the gel. 2. Pour out the buffer. 3. Unscrew the clamps from the sandwiches and remove by using the Wonder Wedge plate separator tool to separate the plates. 4. Carefully lift off the glass plate. Handle the gel with care to avoid damaging it. Label the 1st lane of the gel. Always wet the gel in the buffer (transfer buffer). 5. Rotate the gel in transfer buffer for 15 min at cold room. 6. Prepare the membrane. Wet it gradually in transfer buffer. Also rotate for 15 min at cold room. 7. Clean the unit.

Transfer to the membrane

1. The transfer buffer is made up ahead of time and pre-cooled to 4 C. 2. Cut the membrane slightly larger than the gel. Wet it in transfer buffer slowly and gradually. Mark it. Always use gloves. 3. Rotate the membrane in transfer buffer for 15 min at cold room. 4. Wet two pads and four pieces of filter paper in transfer buffer. 5. Prepare the sandwich as follows: from the bottom to the top (from negative to positive)

One piece of pad Two pieces of filter paper Gel Membrane Two pieces of filter paper One piece of pad

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Note: press out any air bubble. 6. Insert the sandwich into the transfer apparatus. Pay attention to the negative (black) and positive (red). 7. Fill the transfer apparatus with buffer. Pour the buffer slowly to prevent bubble formation. 8. Cool to 4 C and transfer at a constant current (900 mA). Slowly stir the buffer when transfer. 9. Time: about 1 hour. (add 6 min after the used buffer each time). Take care: Not to trap any air bubbles between layers. Collect the transfer buffer to flask for next use.

Primary antibody
1. Wash the membrane in TBST for 6 times (5 min/time). 2. Block the non-specific binding sites by incubating the membrane in 5% BSA (or non-fat milk) in TBST for 1-2 hour (25 ml). 3. Wash the membrane 3 times by l l l TBST, 10 min/time. (or 6 times, 5min/time) 4. Dilute the 1st Ab. (1:2500) into TBST 10 ml (final concentration: 0.5mg/ml, so in 10ml TBST, add 200mg/ml of 1st Ab. 25ul). Rotate to mix it. Wrap it by plastic wrap paper for sealing. Mark the name of the antibody. 5. Incubate it in cold room for overnight by rotating. 6. Collect the antibody (in TBST) to a sterilized tube for the next use. (NOTE: add 2% sodium azide 100ul, the final concentration is 0.02%). Mark it and store at 4 C. 7. Wash the membrane six times by TBST for 10 minutes/time.

Secondary Antibody
1. Dilute the HRP-labeled 2nd Ab. in 10 ml TBST (1:5000 or 1:10,000). 2. Rotate it for 1 hour at RT. 3. Wash 6 times in TBST for 10 minutes each time.

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Enhanced chemiluminescence (ECL) to develop

1. Prepare the Chemiluminescence Reagent by mixing equal volumes from Bottle 1 and Bottle 2 immediately before use. 2. Incubate the reagent with the membrane for one minute at room temperature. 3. To drain excess reagent, hold it vertically and touch it against tissue paper. 4. Place the membrane between the covers of a polypropylene sheet protector with the interleaf removed. Gently smooth out any air pockets. 5. Place the membrane, protein side up, in the film cassette. 6. Switch off the lights and carefully place the film on top of the membrane. 7. Expose the membrane for 30-60 seconds, then develop. 8. Repeat the film exposure, varying the time as needed for optimal detection.