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Assemble the gel apparatus. Carefully unwrap the gels and clamp in place in the gel apparatus: wells must face the centre. Fill the central chamber with 1 x running buffer. Check it is not leaking. Add running buffer to the bottom tray.
Prepare the protein samples for loading. Each student needs to prepare their own set of samples. For each tube of the five tissue samples in the ice box, mix 20 μl protein sample with 20 μl of 2 X loading buffer, keeping the samples on ice. Heat in the hot block for 5 min. Place back on ice for 2 min. Spin down for 2 min. Samples are now ready to load on the gel.
Load the gels. Pipette 5 μl of each of your five prepared samples into five wells on the first gel, and 10 μl of each sample onto the second gel. Another student should pipette their samples onto the same gels. Load one lane on each gel with 4 μl of the marker proteins. Make sure you record which sample you load in each well, in your lab book!
Start the electrophoresis. Check the buffer level in the central chamber is as high as it can go. Place the lid on the gel tank, plug the electrodes into the powerpac. Run at 120-150V (with maximum current) for about 45-50 min. Check that the samples are running the right way before you leave the lab (for lunch). Make sure you record the exact conditions you use.
Dismantle the gels. Allow the gel to run until the bromophenol blue dye front is about 1 cm from the bottom. Turn off the power and remove the gel plates. Place the plates onto paper towels on the bench. Remove the spacers, then use a spacer to carefully prise the plates apart. Take care not to break the glass plates! The gel should remain attached to one plate. Please make sure you place all spacers and plates back into the red trays.
Stain the first gel. With wet gloves carefully lift the gel off the plate and place into a clean white tray. WEARING SAFETY GLASSES add just enough Coomassie staining solution to cover the gel, and leave on the shaking platform at the front of the lab. Do NOT immerse the plate into the stain! Leave the gel in stain for approximately one hour. Meanwhile…
Prepare the second gel for blotting. Place it in a clean tray. Trim off the stacking gel. Soak in transfer buffer for 10 minutes.
Prepare the membrane. Label the nitrocellulose membrane in pencil along the top (long) edge. Include your initials and date so you can identify your blot, and write “TOP” to indicate the gel orientation. Wet this in electroblotting/transfer buffer.
Assemble the blotting sandwich. Dip two pieces of filter paper into blotting buffer to moisten, then place one on top of the other. Place the wetted nitrocellulose membrane on top, with the writing side facing up. With wet gloves, lift your gel out of the transfer buffer and place onto the nitrocellulose, with the top (well edge) of the gel against the edge labelled “top” on the membrane. Use a wet gloved finger to smooth out any air bubbles. Place two more moistened filter papers on top, avoiding air bubbles. Remember to keep your sandwich this way up: gel on top of membrane.
Place blotting sandwich into blotting cassette. Protein is negatively charged and so migrates towards the anode (clear side of the cassette). You must place your sandwich into the cassette so that the membrane is closer to the positive (clear) side, to ensure the proteins migrate from the gel to the membrane! Include a sponge on either side, soaked in transfer buffer.
Blot gel. Place your cassette into the blotting tank, ensuring the black side is nearest the cathode (black). When two cassettes are in the blotting tank, add an ice pack and fill the tank with transfer buffer. Blot at 100V for 45 minutes. Use this time to practice the calculations. After transfer is complete, remove the nitrocellulose membrane (your blot) from the blotting sandwich, rinse with distilled water then leave to air dry overnight. Place the gel (not the blot!) into a clean tray, WEARING SAFETY GLASSES add Coomassie, leave on shaking platform for 15 min.
Destain both gels. WEARING SAFETY GLASSES tip off the stain, rinse once with a little Destain, then cover with Destain. Leave shaking overnight.
IMMUNODETECTION
The aim of Day 2 is to perform immunodetection on the blot, to identify which sample(s) contain the protein of interest, and to determine the approximate molecular weight of the band by comparing it to the marker ladder. You should also destain and image your gels, from yesterday. Use the time during the washes or longer incubations to practice making a standard curve from an image of an unrelated blot.
Block the membrane. Start by making up 20 ml of a 5% solution of MARVEL dried skimmed milk powder in TBST. Only once we have checked your solution is made up correctly should you continue. Wet your blot in TBST, then leave it in the milk solution for 30 minutes, on the shaking platform.
Destain your gels. Return to your gels from yesterday. WEARING SAFETY GLASSES tip the Destain off, into the waste container; cover with fresh Destain and place back on the shaking platform to destain further.
Incubate blot with primary antibody. Tip off the blocking solution and replace with the primary antibody solution. This contains primary antibody diluted 1:20,000 in TBST containing 5% dried skimmed milk powder. Incubate for 60 min on the shaking platform. The primary antibody you will use is a rabbit monoclonal against β-galactosidase. We will have lunch break.
Wash the blot. Tip off the primary antibody solution, rinse the blot in TBST, then perform 3 washes of about 3 minutes each, in TBST. Agitate the blot gently at your bench while washing.
Incubate with secondary antibody. Replace the TBST with the secondary antibody solution. This contains secondary antibody (in this case, goat anti-rabbit AP-conjugated antibody), diluted 1:20,000 in TBST containing 5% milk. Incubate for 45 minutes on the shaking platform. During this time, perform the analysis of another blot image, to practice drawing a standard curve.
Wash the blot. Tip off the secondary antibody solution, rinse the blot in TBST, then wash in TBST 3 times for about 3 minutes each. Gently agitate the blot while washing. While you do this, compare the marker bands to those on the printed sheet, and work out the sizes of the bands you can see. You may not be able to see all of them; use the colours and spacing to help.
Prepare the substrate buffer. During the steps above, make up the substrate buffer for carrying out the chromogenic development. You need to make 20 ml of solution containing 100 mM Tris-HCl, pH 9.5; 100 mM NaCl and 5 mM MgCl2. You will need to do this several times (so both students in a pair can practice!). You will be provided with stock solutions of:
1 M Tris-HCl, pH 9.5 - 5 M NaCl - 1 M MgCl2 . Make sure your recipe is checked by a demonstrator.
Develop the blot. Wash the blot twice for 5 mins each in 20 ml substrate buffer. Replace with 10 ml fresh substrate buffer and add 80 µl NBT/BCIP solution. Protect from the light. Return to the shaking platform. Development may take around 20-30 minutes. Stop the reaction by washing the blot in TBST.
Image gels. WEARING SAFETY GLASSES, tip off the Destain, rinse gels with tap water, and place both gels into a plastic bag. Ensure the bag is wiped completely dry on both sides then take a photo – there is a lightbox available to help with this.
Image your blot. Place your developed blot in a plastic bag. On the bag around the edge of the blot (not over it!), make small marks in fine marker pen to indicate the positions of the marker bands and any immunodetected bands. Take a photo.
Compare your blot and Coomassie-stained gels. Which samples contained the immunodetected band? How does the relative abundance compare between samples? How does the overall protein abundance vary between the different samples? Use the molecular weight markers to work out the apparent Mr of your immunodetected protein. Make sure you’ve recorded which tissue was used to make each sample before you leave the lab.