Western Blotting

Dissolve PM sheets in 100 iL solubilization buffer and transfer entire sample to a microcentrifuge tube using a cell scraper. Determine the total protein content of the sample by performing spectrofluorometric analysis as follows.

1. a. Dilute 10 ^L of the solubilized PM fraction in 60 ^L in solubilization buffer.

b. Mix with 0.25 mL of 0.2 M sodium borate buffer (pH 9.0), and incubate at RT for 5 min.

c. Add 20 ^L of fluorescamine solution while vortexing vigorously.

d. Following a 20-min incubation at RT, measure fluorescence at 395 nm excitation and 460 nm emission using a Shimadzu RF5000U spectrofluorophotome-ter set at high sensitivity. A standard curve should be performed using bovine serum albumin (BSA) in the range from 0.1 to 15 ^g/mL.

2. Fractionate samples using SDS-PAGE.

3. Transfer to polyvinylidene difluoride (PVDF) membranes (Millipore) in transfer buffer for 3-4 amp hours at 4°C.

4. Following transfer, incubate membrane in TBS-0.1% Tween-20 for at least 10 min.

5. Incubate in blocking buffer twice for 30 min each at RT.

6. Rinse three times quickly with TBS-0.1%Tween-20.

7. Incubate with primary antibody solution for 1 h at RT with constant slow agitation. The primary antibody is diluted as recommended by the manufacturer in 2% BSA in TBS-0.1% Tween-20 with 0.02% Sodium azide. After incubation, return antibody to tube for reuse.

8. Perform the following washes at room temperature:

Wash three times quickly with TBS-0.1% Tween-20. Wash three times for 15 min per wash with TBS-0.1% Tween-20.

9. Dilute horseradish peroxidase-conjugated secondary antibodies (Pierce) 1:5,000 in secondary antibody dilution buffer and incubate PVDF membrane for 1 h at RT with constant slow agitation.

10. Perform the following washes at room temperature: Three times quickly with TBS-0.1% Tween-20; Three times for 15 min each with TBS-0.1% Tween-20; Three times quickly with TBS;

Wash once for 5 min with TBS.

11. Carry out ECL reaction for detection of proteins of interest using the enhanced chemiluminescence system (Pierce) according to manufacturer's specifications and quantify by scanning laser densitometry.

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