Procedure:
A. Electrophoretic separation of proteins:
Prior to western blot analysis serum proteins from healthy controls and cancer patients are separated by 10% SDS-PAGE (50 μg per track). 50 μg of extracted protein (dissolved in 25 μL of lamellae buffer) from each subject, is denatured in dry bath at 100°C for 5 min. These proteins are resolved in SDS-PAGE on the basis of their molecular weight. Also run pre-stained protein molecular weight marker for monitoring migration process. For detail protocol of SDS-PAGE users are suggested to follow the Experiment 3.
B. Western blotting:
1. Cut the filter papers (6-8 pieces for each gel) according to the gel size.
2. Cut the membrane without touching its surface and incubate in methanol for 5 min to activate the membrane.
3. Soak all the filter papers and membrane in transfer buffer till use.
Figure 1. Soaking of filter papers and membrane in transfer buffer
4. Add enough amount of transfer buffer on the surface of the blotting apparatus.
Figure 2. Pouring of transfer buffer on the surface of the blotting apparatus
5. In the western blot apparatus, keep 4 filter papers soaked with transfer buffer and slide a test tube gently over each piece of filter paper to remove any trapped air bubbles, and place the membrane, followed by gel with same orientation in electrophoresis. Place 4 more filter paper sheets on the gel soaked with transfer buffer.
FFigure 3. Placement of filter papers and membrane and removal of trapped air bubbles
6. Keep sufficient buffer on the filter paper during running.
7. Proteins separated in 10% SDS-PAGE and then transferred onto PVDF membranes under semidry conditions by using ECL semi-dry transfer unit.
Machine settings:
ü Set current: area of the gel (cm) X 0.8 mA
ü Transfer time = 1 hr to 1.30 hr.
Figure 4. Setting of current for semi-dry transfer during western blotting
Figure 5. Transfer of the proteins separated on SDS-PAGE gel onto PVDF membranes under semidry conditions by using ECL semi-dry transfer unit
8. Once the transfer process is completed, the membrane can be stained with Ponceau. Ponceau staining of the transferred blots containing the resolved proteins can esure equal loading and transfer of the samples in each lane
Figure 6. Ponceau staining of the transferred blot
9. After running, transfer the blot into a small container with 5% skim milk and keep under shaking condition on a rocker for 2-3 hr to block any non-specific bindings on the membrane.
10. Discard the blocking solution and add 3-5 mL (amount depends on the size of the blot) specific primary antibody solution (1:1000 dilution in TBST) and incubate for 2 hrs under shaking condition.
11. After incubation in primary antibody wash the blot in TBST for 3 times (10 min each) by shaking rigorously in shaker.
12. Add 3-5 mL (amount depends on the size of the blot) HRP conjugated secondary antibody solution (1:5000 dilution in TBST) and incubate for 1hr under shaking condition.
13. After incubation, wash the blot with TBST for 6 times (10 min each).
14. Add 60 μL TMB substrate solution (in 3mL TBST) and shake gently for specific amount of time or until desired protein band intensity is achieved. Wash the developed blot with distilled water.
C. Scanning and analysis: After developing, scan the blots using LabScan software version 6.0 (GE Healthcare) and save the image at 300 dpi. in Tiff format. The level of differential expression (fold-change) of different serum proteins in cancer patients compared to the healthy controls can be measured semi-quantitatively by densitometric analysis of the WB images using Image Quant TL (IQTL) software. For detail procedure of densitometric analysis using Image Quant TL (IQTL) software, please see the Experiment 3.
Composition of buffers:
1. Loading buffer or Laemmle buffer (10 mL)
SDS : 2%
Glycerol : 25%
β-mercatoethanol or DTT : 5%
Bromophenol blue : 0.01%
Trsi-HCl (pH 6.8) : 62.5 mM
2. Running buffer (10X)- 1 Lit
Tris-HCl : 25 mM (30.3 g)
Glycine : 192 mM (144 g)
SDS : 0.1 % (10 g)
3. Composition of resolving gel 10% (6 mL)
Acrylamide-bisacrylamide mix (29:1) : 1.7 mL
1.5 M Tris-HCl (pH 8.8) : 1.3 mL
10% SDS : 50 μL
10% APS : 50 μL
TEMED : 3 μL
Water : 1.9 mL
4. Composition of Stacking gel (5%)
Acrylamide-bisacrylamide mix (29:1) : 500 μL
0.5 M Tris-HCl (pH 6.8) : 375 μL
10% SDS : 30 μL
10% APS : 30 μL
TEMED : 3 μL
Water : 2.05 mL
5. Transfer buffer for 2 Lit
Glycine : 28.8 g
Tris-base : 6.06 g
Methanol : 400 mL
6. TBST buffer (pH 7.6) for 1 Lit
NaCl : 8 g
KCl : 0.2 g
Tris base : 3 g
Tween 20 : 0.6 mL