Western Blotting Method

Materials and Method:

The COSH form for the western blotting method is added into the appendix.
There was no ethic consideration to take into account.

    • Large and small tubes.
    • Cassettes
    • Combs
    • Measuring scale
    • Gel tank
    • Knife
    • Scissors
    • Tweezers
    • Conical flasks
    • Beakers
    • Membrane paper
    • Plastic box
    • Tray
    • Sponges
    • Fuse paper
    • Lamp
    • Films
    • X-ray film cassette
    • Pipettes
    • Test tube rack

    • Water
    • 30% acrylamide mix
    • 1.5 m Tris (pH 8.8)
    •   10% SDS buffer
    • Ammonium persulfate
    • TEMED
    • Butanol
    • 10x tri/glycerine/SDS buffer
    • Tween
    • Human stem cell samples, 1-5
    • TGF and PDGF samples
    • Molecular weight marker
    • Empty sample marker
    • Strip buffer
    • 2-mercaptsethanol
    • Milk powder
    • Developer and fixation solution


Prepare resolving gels for tris-glycine SDS-Polyacrylamide Gel Electrophoresis:

Two solutions, 10% resolving and 6% stacking gel were prepared to make the gel.
25ml of 10% resolving gel was made by adding 9.9ml of water, 8.3ml of 30% acrylamide mix, 6.3ml of 1.5 m Tris (pH 8.8), 0.25ml of 10% SDS, 0.25ml of 10% ammonium persulfate and finally 0.01ml of TEMED was added. About 10ml was then poured into each cassette until the second line using a large pipette.   Immediately after about 300-400µl of butanol was added on top of the resolving gel. The cassettes were then left for the gel to set.

15ml of 6 % stacking gel was prepared by adding 7.9ml of water, 3ml of 30% acrylamide mix, 3.8ml of 1.5 m Tris (pH 8.8), 0.15ml of 10% SDS, 0.15ml of 10% ammonium persulfate and 0.012ml of TEMED. When the resolving gel in the cassette was formed any left over butanol was poured out. The stacking gel was then added to the cassette on top of the resolving gel and the combs were added on top of cassettes and left...