Western Blot method.

Western blotting technique consists of the following parts:
1)separation of polypeptides by SDS-PAGE;
2)electrotransfer of separated proteins from the gel into the blotting paper;
3)labeling of the transferred proteins by antibodies conjugated with the enzyme
4)detection of the labeling enzyme signal.
1. SDS-PAGE electrophoresis.
10x running buffer:
30 g of Tris base
140 g of glycine
10 g of SDS, all diluted in
1L of bidistilled water
Precool before use		 2x solubilization buffer:
60 mM Tris-HCl, pH 6.8
20% SDS
0.0004% bromophenol blue
10%_-mercaptoethanol
20% glycerol
Stacking gel buffer:
0.5 M Tris-HCL, pH 6.8
0.4% SDS		 Separation gel buffer:
1.5 M Tris-HCL, pH 8.8
0.4% SDS
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 To prepare 10% separation gel, mix 2.5 ml of 40% acrylamide/bis (9:1) solution, 2.5 ml of separation gel buffer, 5 ml of bidistilled water, 50 µl of ammonium persulfate and 5 µl of TEMED. Cover the polymerizing gel by the layer of distilled water as it solidifies in the rig.
To prepare stacking gel mix 1 ml of 40 % acrylamide/bis (9:1) solution, 2.5 ml of stacking gel buffer, 6.5 ml of bidistilled water, 50 µl of ammonium persulfate and 10 µl of TEMED. Dump out the layer of distilled water and layer the stacking gel on top of the separating gel.
To prepare sample homogenize the particular material in solubilization buffer and incubate 30 min-1 hour at 40ºC. Centrifuge sample for 2 min at 12000g.
Start electrophoresis in the precooled running buffer at 70 v until the sample enter the separating gel, and then increase voltage up to 100 v.
2. Electrotransfer of the separated polypeptides into the blotting paper.
10x transfer buffer:
30.3 g Tris base
144 g glycine, all diluted in
1 L distilled water
Adjust pH with HCl to 8.3
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 Soak the blotting paper cut to the size of the separating gel (in our lab we are using Immobilon P) in methanol for 2 min, and then accurately put the paper on the surface of water for 10 min. During this procedure the water replaces methanol from the paper pores.
Mix 100 ml of 10x transfer buffer with 850 ml of bidistilled water, cool the solution in the cold room and add 50 ml of metanol.
Cut off the stacking gel and soak the separating gel in the prepared solution for 10 min at 4ºC. Soak 2 pieces of the Watman 3MM paper with a size of separation gel in the prepared solution for 2 min. Assemble sandwich, remove bubbles between gel and Immobilon P and perform electrotransfer in the prepared solution for 1 hour at 100v.
3. Labeling of the transferred proteins with antibodies.
10x TBS-T buffer:
24.2 g Tris base
80 g of NaCl, pH 7.5
10 mls of Tween-20, all diluted in
1L of bidistilled water
Store at 4ºC.
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 Prepare 100 ml of TBS-T buffer, add 5g of dry nonfat milk. Shake vigorously. Incubate the blotting paper in 15 ml of the prepared milk solution for 1 hour at room temperature. Wash the blotting paper two times with 15 ml of TBS-T buffer for 5 min.
Dissolve the primary antibodies in the 15 ml of milk TBS-T buffer and add to the blotting paper. Incubate 1 hour at room temperature.
Wash the paper with 15 ml of TBS-T buffer 4 times for 5 min.
Dissolve the secondary antibodies (in our lab we are using antibodies conjugated with the horseradish peroxidase) in 15 ml of milk TBS-T buffer and add to the blotting paper.
Incubate 1 hour at room temperature.
Wash the paper with 15 ml of TBS-T buffer 4 times for 5 min.
4. Detection of enzyme activity on the blotting paper.
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 Prepare 6 ml of the substrate solution for horseradish peroxidase in accordance with the Amersham ECL kit protocol.
Incubate the blotting paper with this solution for 1 min.
Wrap the wet paper with the Cling Wrap, put the Kodak X-OMAT AR film on the surface of the covered blotting paper and detect chemiluminescence.