To start, prepare the four sheets of filter paper, each measuring 12.5 by 7.5 centimeters. Stack two sheets together and place the stack in transfer buffer to soak through. Place the PVDF membrane in 95%ethanol and agitate the membrane on a rocker for one minute.
Recollect the ethanol. Immerse the membrane in transfer buffer. And agitate for two minutes.
Next, lay the soaking filter paper stack on a flat, movable surface, such as a large lid and flatten it using a roller. Using a gel releaser or tweezers, carefully position the PVDF membrane on the stack. Afterward, place a single sheet of dry filter paper on this membrane and glide the roller over the paper to absorb any excess buffer that's on the membrane's surface.
Lift off the top filter paper without rubbing against the membrane. Take isolated fiber samples from the freezer and thaw them at room temperature before centrifuging and thoroughly re-suspending the sample. Place a one microliter droplet of each sample in the center of the designated membrane area.
Avoid touching the membrane with the pipette tip. Allow the sample droplets to absorb entirely into the membrane for 15 minutes. Then using a gel releaser or tweezers, carefully lift the membrane from the damp filter paper stack.
Place it onto a dry sheet of filter paper and leave it for at least five minutes to dehydrate. Reactivate the membrane after the sample spots have turned completely white. Then proceed with immunolabeling the target protein.
Learn how to use the signal panel intensity to classify the intensity of the MHC isoforms and Actin signals from the dot blot images. Strong, medium and little target protein detection is shown by saturated, moderate and faint signals respectively. For fiber type identification, initially compare the myosin heavy chain IIA and myosin heavy chain one results.
Document the fibers that display only a single myosin heavy chain isoform with either saturated or moderate signal intensity. Record the fibers detected with Actin and no detection of myosin heavy chain one or IIA as a potential type 2X. If a faint myosin heavy chain isoform signal is present with a moderate desaturated Actin signal, record it as unidentified.
Discard samples with faint or undetected target proteins. Fibers displaying saturated or moderate signals of both MHCIIA and MHC1 should not be used for fiber type-specific preparation. After MYO1D ID, prepare type one and two samples by combining fibers with saturated or moderate signals for the respective MHC isoform.
Use 10 microliters of each fiber type-specific sample and separate them on a precast gel by SDS page, following the manufacturer's guidelines. Use a gel imager to detect the target MHC isoform, following the manufacturer's protocol. Compare the signal intensity of each MHC isoform in all fiber type-specific samples.
Fiber type ID is confirmed by the correct MHC isoform at moderate or saturated signal intensity. Immunolabeling of the dot blot allowed the identification of type two fibers, type one fibers and potential type 2X fibers. The fiber type-specific samples were validated, using western blotting and myosin heavy chain specific antibodies.
