Biol 307

Cell and molecular biology

Western Blotting

I. Transfer

1. Soak gel and PVDF membrane in transfer buffer for 10 min before setting up RioRad apparatus.

2. Transfer O/N at 30 V (90 mA) or 4-6 h at 70 V (can reduce buffer strength to reduce heat).

II. Antibody binding

1. Wash 2X 10 min in TBS.

2. Block 1 h in 0.2% non-fat dry milk in TBS.

3. Wash 10 min in TTBS

4. Add Primary antibody in TTBS + 0.2% milk. Use a 1:10,000 dilution for GFP or LDH Ab. Incubate 3 hours, can go O/N

5. Wash 4 x 10 min with TTBS.

6. Add Secondary Ab, (5 mL in 100 ml TTBS + 0.2% milk). Incubate 1 h.

7. Wash 4 x 10 min with TTBS.

8. Add substrate, 5 min.

9. Detect by imaging, no light, open iris, no filter.

III. Buffers

Transfer buffer

25 mM Tris, pH 8.3, 192 mM Glycine, 10% methanol.

2X buffer: 6.06 g Tris, 28.8 g Glycine, 200 ml methanol, water to 1 L

TBS

20 mM Tris 7.5, 500 mM NaCl.

10X TBS: 40 ml 1 M Tris 7.5, 58.44 g NaCl, water to 200 ml.

TTBS

TBS with 0.1% Tween 20.

1 ml Tween 20 in 1 L TBS.

TTBS + milk

TTBS with 0.2% non fat dry milk.

0.2 g dry milk in 100 ml TTBS.

IV. Antibodies and antigens

GFP

Primary, rabbit polyclonal IgG; secondary, goat anti rabbit-alkaline phosphatase, detect with BioRad luminescence substrate (Immunostar)

LDH

Primary, goat polyclonal IgG; secondary, secondary rabbi anti goat-horseradish peroxidase, detect with BioRad luminescence substrate (Immunstar WesternC).