1.Preparing the sample:
-Cutting 20 um frozen sections to a slide, 3-5 of them; depends on the tissue size.
(The slides can store at –80C for later study)
-Air dry for 5 min or so.
-Dissolution:
Triple-detergent lysis buffer (from Sambrook Fritsch Maniatis):
50 mM Tris-HCL
150 mM NACL
0.1% SDS
100ug/ml PMSF
1ug/ml Aprorinin
1% NP-40
-100-200ul of lysis buffer is added to each slide, mix tissue with pipette tips
-Scrape the mixture all from slide to a 1.5 ml tube.
-Vortex well, put in to ice for 30-60 min
-Spin 10 min at 14K rpm -Take the supernatant into a new tube
2. SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis):
-Choose the percentage of SDS-Polyacrylamide Gel according to protein molecular size.
Acrylamide concentration (%) Linear range of separation (KD)
- 12-43
- 16-68
- 36-94
- 57-212
-Add 10-20ul of sample to same amount of 2X loading buffer (1:1), mix well, and include Protein Marker.
2X Loading buffer:
100 mM Tris-HCL (pH6.8)
200 mM DTT
4% SDS
0.2% Bromophenol blue
20% glycerol
(DTT should be added just before the buffer is used, from 1M stock)
-Boiling for 5-10 min
-Preparing the GEL: mount the gel in the electrophoresis apparatus.
-Add Tris-glycine electrophoresis running buffer to the top and bottom reservoirs.
Tris-glycine Running Buffer:
25 mM Tris
250 mM glycine (electrophoresis grade) (pH8.3)
0.1% SDS
-Remove any bubbles
-Loading up 20-40ul of each sample in a predetermined order into the bottom of wells.
-Attach the electrophoresis apparatus to an electric power supply (the positive elecrode should be connected to the bottom buffer reservoir)
-Running at 200V for 45’-60’
-Remove the glass plates from the electrophoresis apparatus and place them on a paper towel. Using a spatula, pry the plates apart.
-Mark the orientation of the gel by cutting a corner from the bottom of the gel.
-Remove the gel from the electrophoresis apparatus and incubate it in Western
Transfer Buffer for approximately 10 min to remove detergent.
3. Blotting Transfer:
(Caution: Always wear gloves when handling the gel or membrane. Maneuver the gel with forceps or gloved hands to minimize background)
-Fill the Bio-Ice cooling unit with water and store it in –20C until ready to use. After use, return the cooling unit to the freezer for storage.
-Prepare Western Transfer Buffer:
Transfer buffer:
10% methanol,
24 mM Tris
194 mM glycine
in advance and refrigerate.
(Caution: The buffer must be at 4°C for electrophoretic transfer.)
-Cut the membrane and filter paper to the dimensions of the gel.
-Equilibrate the gel and soak the member, filter paper, and fiber pads in transfer buffer for 15 min.
-Prepare the gel sandwich:
1) Place the cassette, with the gray side down, on a lean surface.
2) Place one pre-wetted fiber pad on the gray side of cassette.
3) Place a sheet of filter paper on the fiber pad.
4) Place the equilibrated gel on the filter paper.
5) Place the pre-wetted membrane on the gel
6) Complete the sandwich by placing a piece of filter paper on the membrane.
(Removing any air bubbles, which may have formed; it is very important for good results. Using a glass tube to gently roll air bubbles out)
7) Add the last fiber pad.
8) Close the cassette firmly, being careful not to move the gel and filter paper sandwich. Lock the cassette closed with the white latch.
-Place the cassette in module. Repeat for the other cassette if you have one more.
-Add the frozen Bio-Ice cooling unit. Place in tank and completely fill the rank with buffer.
-Add s standard stir bar to help maintain even buffer temperature and ion distribution in the tank. Set speed as far as possible to keep ion distribution even. (Optional)
-Put on the lip; plug the cable into the power supply.
-Transfer conditions: (always using the Bio-Ice cooling unit)
Overnight transfer 1 Hour transfer
30V 100V
90mA 350mA
-After the transfer, unclamp the blot sandwich and remove the sheets of blotting paper, exposing the blot membrane. Mark the side of the membrane that was facing the gel. Mark the position of the pre-stained markers, since they may fade away during detection.
4.Detecting the protein specific antibodies:
-Blocking the membrane with 5-10% nonfat dried milk in PBS for 1 hour or overnight at 4C with shaking.
-Wash in PBS with 0.1% Tween20, 3 X, and 10 min for each time.
-Incubate with Primary Antibody in 1%BSA in PBS for 1 hour at room temperature or overnight at 4C by shaking. (Optimal antibody concentration must be determined experimentally; 1:500dilution is good starting point.)
-Wash in PBS with 0.1%Tween20, 3 X, and 10 min of each time
-Incubate with secondary antibody (peroxidase-conjugated goat anti-mouse IgG, etc.) following the manufacturer’s instruction, usually 1:200 to 1:2000; 1 hour at room temperature
– Wash in PBS with 0.1%Tween20, 3 X, and 10 min of each time
5. Developing: (Use of chromomeric substrate- 3,3’-diaminobenzidine, DAB)
(DAB is most sensitive for immunocoupled horseradish peroxidase. It is converted in situ a brown precipitate. It is essential to monitor the course of the reaction very carefully. The reaction should be stopped as soon as the specifically stained bands are clearly visible.)
-Transfer the membrane to a shallow tray.
-Add 10ul H2O2 (30%) to 10 ml of 0.05%DAB in PBS, mix well immediately;
-Pour the DAB to the membrane, incubate at room temperature with gentle shaking in the dark if possible.
-Monitor the progress of the reaction carefully. When the bands are of the desired intensity (2-5 min), wash the filter briefly in water, and in PBS.
-Dry the membrane and photograph it to provide a permanent record of the experiment.
