The overall goal of this western blotting technique for high-molecular-weight glycoproteins is to electrophoretically separate mucins contained in mucus-rich samples. This method can help answer key questions in the field of mucin biology, such as, investigating the polymeric state in relative concentrations of specific mucins harvested from various mucosal surfaces. The main advantage of this technique is that it is easy to establish, relatively low-cost, and requires minimal laboratory equipment.
Demonstrating this procedure will be Sophia Samir, research specialist from my laboratory. Begin the experiment by preparing the gel. Pour 150 milliliters of 1x TAE 0.1%SDS buffer into a microwaveable Erlenmeyer flask to make a six-millimeter-thick gel and add 1.2 grams of agarose powder.
Microwave the flask for 30-second intervals with intermittent swirling until the agarose is completely dissolved. Allow the agarose solution to cool down for up to 10 minutes. Next, place a 10 by 15 centimeter electrophoresis casting tray into a gel caster.
Slowly pour the agarose solution and place the combs into position. Remove any bubbles that may have formed with a pipette tip, then allow the gel to cool and solidify. Once the gel has completely solidified, remove the comb slowly to prevent the wells from collapsing and remove the tray from the gel caster.
Place the gel into the gel box and fill the electrophoresis unit until the gel is fully covered. Add 10%of loading dye to each sample. Homogenize the samples by pipetting up and down.
Then, briefly spin the tubes at 5000 RPM to collect droplets. Next, use positive displacement pipettes to facilitate loading the highly viscous samples. Slowly aspirate 80 to 90 percent of the sample to avoid pipetting bubbles.
Slowly load from the bottom of the well and progressively move the pipette tip upward. For samples that remain attached to the pipette tip, dispense at a 45-degree angle and elongate straight above the well or gently use the side of the well to break the stringy mucus. Accurately loading the viscoelastic samples can be challenging as mucus inherently adheres to the loading tips.
Also, the buoyant force applied by the buffer on to mucus may displace total or part of the loaded volume out of the wells. Run the gel at 80 volts for 60 to 75 minutes for a double-comb gel to prevent overlap between the two rows. After the gel has run an adequate length, turn the generator power off and disconnect the electrodes from the power source.
Carefully remove the gel from the gel box with one hand on either side of the casting tray to ensure the gel remains in place. Carefully place the gel flat into a glass container. Then, rinse the gel with distilled water to remove traces of TAE SDS buffer.
Soak the gel with previously prepared DTT 4x SSC buffer and cover the gel. Next, gently rock the gel at 10 rotations per minute. After rocking for 20 minutes, rinse the gel with DTT-free 4x SSC buffer.
Prepare the vacuum blotter for transfer by carefully cutting the nitrocellulose membrane slightly wider than the gel. Wet a porous mat with distilled water and place it smooth-side up in the blotter. Wet and place two layers of Whatman paper at the center of the porous mat.
Wet a nitrocellulose membrane with DTT-free 4x SSC buffer and place it on top of the Whatman paper. Cover the porous mat with a rubber gasket leaving the opening of the gasket precisely aligned with the nitrocellulose membrane. If the porous mat is still visible, carefully adjust the membrane to ensure no gap remains between the gasket and the membrane.
Wet the nitrocellulose membrane with DTT-free SSC media. Carefully place the agarose gel on top of the membrane. Ensure the gel forms a tight seal with the gasket and the wells of the gel are positioned on the membrane for transfer.
Throughout protein transfer, make sure you maintain a tight seal between the agarose gel and the nitrocellulose membrane. Once the gel is placed on to the membrane, do not move or adjust the gel and keep the gel well hydrated. To minimize the formation of bubbles, squirt a few drops of 4x SSC buffer on the membrane and slide the gel from top to bottom to flush any formed bubbles.
Carefully cover the gel with 4x SSC buffer. Start the mucin transfer by turning on the vacuum blotter for 90 minutes. Add a few drops of 4x SSC buffer on the gel every five to 10 minutes to prevent the gel from drying out.
Upon completion, mark the wells with a pencil for orientation. Discard the gel and retrieve the nitrocellulose membrane with plastic tweezers by the edge of the membrane. Rinse the membrane immediately with 1x phosphate buffered saline or PBS.
Immerse the membrane in 15 milliliters of 3%milk PBS-blocking buffer and place it on a rocker at room temperature for one hour. After incubation, discard the blocking buffer. Next, immerse the membrane in 1%milk PBS primary antibody solution and incubate the membrane overnight on a rocker at four degrees Celsius.
After incubation, discard the primary antibody solution. Then, wash the membrane with PBS while rocking. Transfer the membrane to a nontransparent container and immerse the membrane in 1%milk PBS secondary antibody solution and incubate the membrane at room temperature for one hour on a rocker.
After incubation, discard the secondary antibody solution. Wash the membrane three times with PBS for five minutes each. Finally, rinse the membrane with distilled water to remove salt.
This western blot displays the upregulation of Muc5ac and Muc5b proteins in bronchoalveolar lavage fluids of IL-13-challenged mice compared to control mice. A quantitative analysis shows that the upregulation was 5.1 fold over PBS treated mice. Mucin agarose gel electrophoresis was used to show the reduction of mucin polymers into monomers following treatment with increasing concentrations of DTT.
While attempting this procedure, it is important to remember to confirm antibody specificity before studying a particular mucin. For pharmacological testing, it is important to examine the impact that chemical reagents may have on epitope recognition. After watching this video, you should have a good understanding of how to manipulate viscoelastic samples and determine the polymeric state of mucins and their relative quantification.
