General Protocol - Western Blotting
1. Mix 25-100 µg of total protein from the cell lysate (depends on the abundance of the target protein and the size of the well) with 6X gel loading buffer, boil for 5 min.
2. Separate the protein by SDS-PAGE (acrylamide:bis-acrylamide = 29:1).
3. Transfer the protein to a nitrocellulose membrane. Usually 400 mA, 1 hr at 4°C gives the best results for protein size smaller than 100 kD. For large protein (>200 kD), we suggest to transfer overnight at 50 mA at 4°C.
4. Block the membrane with blocking buffer for 1 hr at room temp.
5. Dilute the antibody in blocking buffer at an appropriate conc. (we suggest to start with 1:500-1000 dilution) and apply to the blot (about 100 µl per cm2).
6. Incubate the blot on a shaking platform for 1~2 hour at room temperature.
7. Wash the blot 3-4 times with PBST, 10 min each.
8. Make appropriate dilution of the HRP-conjugated anti-mouse antibody in PBST, apply to the blot and incubate on a shaking platform for 30 min-1 hour at room temperature.
9. Wash the blot 3-4 times with PBST, 10 min each.
Visualize the target protein by standard ECL® protocol (Amersham)
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