1.

Cultured cells were harvested by centrifugation and were washed twice with PCB,
2. Cells were then resuspended in lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl,
0.1% triton X 100, 4 mM EDTA, 1 mM DTT, 50 µg/mL PMSF, 50 mM NaF, 0.2 % NP-
40 and protease inhibitor cocktail, and were lysed by repeated vortexing (6-7 times) at an
interval of 10 min on ice,
3. The lysed cell suspension was allowed to stand for 30 min at 4oC and centrifuged at 750 g
for 15 min to yield the cell lysates,
4. The protein concentration in the cell lysates was measured by using Bradford reagent.
Equal amount of protein was loaded onto the 10 % SDS-PAGE,
5. By wearing gloves, setup the transfer apparatus and apply a current of 0.65 mA/sq.cm of
the gel for a period of 1.5-2 h,
6. Turn off the electric current and disconnect the leads and the transfer apparatus from the
main power supply. Stain the gel with coomassie brilliant blue to check whether the
electrophoretic transfer is complete,
7. Allow the filter to dry for 30-60 min at room temperature,
8. Cut off the bottom left hand corner of the filter,
9. Block the non-specific binding sites for Immunoglobulin on the nitrocellulose filter by
adding 0.1 mL of the blocking solution per sq.cm of the filter and incubate for 1-2 h at
room temperature with gentle agitation,
10. Discard the blocking solution and immediately incubate the filter with an antibody directed
against the target protein,
11. To the plastic bag containing the nitrocellulose filter, add 0.1 mL of the blocking solution
and an appropriate quantity of the primary antibody,
12. Seal the bag and incubate the filter overnight at 4 oC on a gel rocker,
13. After the incubation is complete, discard the blocking solution and the primary antibody
and wish the filter 3 times with 100-200 mL of PBS,
14. Transfer the filter into a tray containing 200 mL of 150 mM NaCl and 50 mM Tris.Cl pH
7.5. Incubate the filter for 10 min at room temperature with gentle agitation,
15. Transfer the filter to a heat-sealable plastic bag containing 0.1 mL of phosphate-free, azide
free, blocking solution per sq.cm of the filter,
16. Add the enzyme-coupled secondary antibody according to the manufacturer’s instructions
and seal the bag. Incubate it for 2 hours at room temperature with gentle agitation,
17. Transfer the filter to a tray containing 200 mL of 150 mM Tris.CL pH 7.5. Incubate the
filter for 10 min at room temperature with gentle agitation. Repeat this step three more
times,
18. Transfer the washed nitrocellulose buffer to a shallow tray and add 0.1 mL of chromogenic
substrate mixture per sq.cm of the filter. Incubate the filter at room temperature with gentle
agitation,
19. Monitor the progress of the reaction carefully when the bands of desired intensity start
appearing. Dry the membrane in the hot air incubator at 37 oC to fix the color,
20. Take the photograph of the filter.
Transfer buffer for 2.0 litres
Glycine: 28.8 g
Tris: 6.06 g
Methanol: 400 mL

TBST for 1.0 litre (pH 7.6 )

NaCl: 8.0 g
KCl: 0.2 g
Tris: 3.0 g
Tween 20: 0.6 mL
Alkaline Phosphatase buffer for 100 mL (pH 9.5)
NaCl: 0.585
MgCl2: 0.102 g
Tris HCl: 1.11 g