Analysis of Gene and Protein Expression in Atherosclerotic Mouse Aorta by Western Blot and Quantitative Real-Time PCR

Resolving gel (%)

7.5

Total vol

8 ml

ddH₂O

4.5 ml

3.8 ml

3.2 ml

2.6 ml

1.8 ml

30 % Acry/Bis

1.33 ml

2 ml

2.67 ml

3.2 ml

4 ml

1.5 M Tris–HCl (pH 8.8)

2 ml

20 % SDS

40 μL

10 % APS

80 μL

TEMED

8 μL

Mr (kDa)

25–200

15–70

12–45

Stacking gel (%)

Total vol

5 ml

ddH₂O

30 % Acry/bis

0.67 ml

0.5 M Tris pH 6.8

1.25 ml

20 % SDS

25 μL

10 % APS

50 μL

TEMED

5 μL

Carefully cover the top of the gel with water-saturated isobutanol (or any other tertiary amyl alcohol), and allow complete gel polymerization (30–60 min) (see Note 16 ).

Meanwhile, prepare 4 % stacking solution (recipe in Table 1).

Remove the isobutanol top layer and rinse the gel extensively with distilled water.

Dry the inner sides of the glass plates thoroughly with Whatman 3MM paper.

Add 10 μL of TEMED to the stacking gel solution; mix and pour immediately.

Insert the desired clean comb (1 mm thickness), and allow the gel to polymerize completely (approximately 20 min). Check for leaks.

Remove the comb and rinse extensively with distilled water (see Note 17 ).

10.

Place the gel sandwich in the appropriate electrophoresis apparatus.

11.

Fill the tank and cassette holder reservoir with 1× SDS Running Buffer. Ensure that there are no leaks and remove bubbles.

12.

Boil samples at 95 °C in 1× final SDS Sample Buffer for 4–5 min and briefly spin down.

13.

Load samples (~20 μg/lane), including one well with prestained molecular weight markers.

14.

Run gels at 90 V (8–15 milliamps current) until the dye front has moved through the stacking gel. The current can then be increased (25–35 milliamps), and the gel run until the dye front reaches the bottom of the gel.

15.

Remove the glass plates and use the gel for western blot (see Note 18 ).

3.3 Western Blotting

Transfer the proteins from the 1 mm thick gel to a PVDF membrane by using the iBlot^® Gel dry transfer system which allows ultra-rapid protein transfer in 7 min (P3 program) (see Notes 19 – 22 ).

Immerse membrane in blocking buffer and incubate with gentle agitation for 2 h at room temperature or overnight at 4 °C (see Note 23 ).

Wash membrane three times in abundant volumes of TBS-T for 5 min each time, rocking (see Note 24 ).

Add diluted appropriately primary antibody in blocking buffer and incubate overnight at 4 °C (unless otherwise specified), with gentle agitation (see Notes 25 and 26 ).

Wash three times in TBS-T for 5 min each time.

Dilute secondary antibody in blocking buffer (as recommended by the manufacturer), and incubate with the membrane for 1 h at room temperature with gentle agitation.

Wash three times for 5 min each with an excess of TBS-T.

Detect proteins with a chemiluminescence kit according to the manufacturer’s recommendations.

3.4 Membrane Stripping

If samples are limited, bound antibody can be stripped from membranes, which can then be reprobed with a different first antibody.

Immerse the membrane in Stripping Buffer and incubate at 50 °C for 30–60 min (see Note 27 ).

Wash membranes extensively with TBS-T at least five times for 5 min each time.

Block membranes in the appropriate blocking buffer based on the requirements of your first antibody (see Note 23 ).

3.5 RNA Isolation

Disrupt mouse aortic tissue (maximum 100 mg of tissue) in sterilized screw-cap RNAase-free tubes with 700 μL of phenol-based RNA Lysis Reagent (e.g., QIAzol) for 2 min at 50 Hz speed (CAUTION! See Notes 28 – 31 ).

Repeat the process until complete tissue disruption is achieved (see Notes 7 and 8 ).

Transfer homogenate to a new tube and leave for 5 min at room temperature.

Add 140 μL (1/5 v:v) chloroform and shake vigorously for 15 s or until a uniform pink suspension is obtained.

Leave for 2 min at room temperature until phase separation is achieved (see Note 32 ).

Centrifuge at 12,000 × g for 15 min at 4 °C.

Transfer the RNA-containing upper aqueous phase to a new tube.

Only gold members can continue reading. Log In or Register to continue