Western_Blotting_Method

Materials and Method: Safety: The COSH form for the western blotting method is added into the appendix. There was no ethic consideration to take into account. Equipment: • 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 Reagents: • 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 Method: 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 for about 30 minutes to form. Preparing samples to be loaded: Tubes 1 to 5 contained human stem cells samples. Each sample had a different time course as shown below: • Sample1 = 0 hours • Sample 2 = 4 hours • Sample 3 = 12 hours • Sample 4 = 24 hours • Sample 5 = 24 hour + force loading. Also there were samples of TGF (concentration 5mg/ml) and PDGF (concentration 5mg/ml) (roughly 1µl of each). 100µl of SDS buffer was added in to each tube of TGF and PDGF. The tubes were then centrifuged for 10-20 seconds. Then all samples were placed on to the hot plate at 95∙c and heated for about 5-10 minutes. After heating the samples were then centrifuged and left to cool down at room temperature. Preparing the gel tank and loading the samples: The cassettes were labelled from 1 onwards and a small dot was placed on the cassette were each well was. The tape at the bottom of the cassette was removed with a sharp knife. The cassette was then placed into the gel tank and run buffer (100ml of 10x tri-glycerine/ SDA buffer diluted into 1:10) was added into the gel tank. Then the combs on the cassettes were slowly removed and more run buffer was then added to the tank. 15µl of human stem cell, TGF, PDGF (concentration 5mg/ml) and molecular marker (MW) samples were then loaded into each well (as shown below). The empty wells were loaded with 10µl of empty sample buffers. The lid to the gel tank was then placed and the electrophoresis machine was set to 80V for 30 minutes. Then to 150- 200V for 90 minutes roughly. Electroblotting: Electrophoresis machine was stopped and the cassettes were removed. The cassettes were opened and the gels were added on the membrane paper and left to soak in the transfer buffer. Next the sponges were soaked in the transfer buffer (20ml of Tris-glycine transfer buffer (25x) added to 50ml of 10% methanol of 500ml) and placed on to electrophoresis equipment. Fuse paper was soaked and placed on top of the sponge, then membrane paper and the gel was placed on top of the fuse paper and another fuse paper and sponge was layered on top. Then it was covered with the lid and placed into the gel tank. Into the middle of the tank transfer buffer was added and to the sides water was added. The electrophoresis machine was set to 220 mA for 2 hours. Once the electrophoresis machine had stopped the membrane paper was removed using a tweezers and each membrane papers were placed into small trays containing 5% milk. Trays were placed on to the see-saw rock for 30 minutes at 30 osc/min. Preparing first antibody: The antibodies were diluted by 1:1000 by adding 10ml of wash buffer, 1ml of water and 10µl of the antibody. When the orbital shaker stopped the milk from each tray was poured out and each antibody was added into the according trays. The trays were then covered and taken to the cold room and placed on the platform rocked for over night incubation (24hours). Washing the membrane paper: The first antibody solution was removed and the membrane paper was washed for 2 minutes with wash buffer (40ml of 50x Tris- buffered saline added to 2l of water and 1ml of tween 20) on the see-saw rock at 60 osc/min and then for 10 minutes at 60 osc/min, this was repeated 3 times. Preparing the second antibody: 10ml of wash buffer was added into each tube and 10µl of anti-mouse IgG, HRP linked antibody was added into one tube and 10µl of anti-rabbit IgG, HRP-linked antibody was added into second tube. Once the see-saw rocker had stopped the second antibody was added into the correct trays. The see-saw rocker was then set for 30 minutes at 60 osc/min. Then the membrane paper was washed again with wash buffer for 2 minutes then for 10 minutes and repeated 3 times. Immunodetection: The membrane paper was removed and dried on tissue paper and placed in a plastic box filled with wash buffer. 3ml of detection reagent 1 and 3ml of detection reagent 2 was added together in a small tube. The membrane paper was picked up using tweezers and dried on paper and placed onto cling film. Then the detection reagents were added onto the membrane paper and then the membrane paper was dried and placed on a plastic pocket which was placed inside the x-ray film cassette. The sides of the plastic sheet were taped down. Next the developer and fixation solution was added into two trays (half full) in the darkroom and one tray was filled with cold water. The films were then taken out and cut in half and placed on to the X-ray film cassette for about 60 seconds. The film was then removed and placed into the developer solution tray. This was left for 3 minutes and then rinsed in water and placed in the fixation tray for 3 minutes. The films were then left in cold water for about 10 minutes to wash off and then left to dry. The above steps were repeated for each antibody. The antibodies used were: • P-ERK • ERK • α-SMA • CCN-2 • Integrin β1 • Moesin • Integrin β5 • Collagen • Syndecan- 4 • GAPDH • Fibronetin (FN) Stripping and reprobing membranes: After the developing procedure the membrane paper was reused for new antibodies by removing the primary and secondary antibodies. The membrane paper was submerging in strip buffer (50ml of strip buffer added to 50ml of water and 0.7ml of 2-mercaptsethanol). Membrane paper was placed into a water bath at 50 .c for 30 minutes. After this the membrane paper was washed with wash buffer on the see-saw rocker at 60 osc/min and this was repeated 3 times for 10 minutes. After this 5% milk was added into small plastic trays and membrane paper added into each tray and placed on the see-saw rocked for 30 minutes at 30 osc/min. The same method from above was repeated from this point onwards. ----------------------- 1 2 3 4 5 X TGF PDGF X MW