Western Blotting

Western Blotting is used extensively as an analytical method to detect proteins.  Oxford Biomedical Research supplies hundreds of antibodies that are optimized for Western Blotting. The protocol below is an example Western Blotting as is the PDF file.

1. Perform SDS-Page
2. Cut one nitrocellulose paper and two pieces of filter paper slightly larger than gel to be transferred. 
3. Immerse open clamp assembly (or equivalent) in a shallow bath of cold transfer buffer.  Orientate sponges, filter papers, nitrocellulose paper and gel and allow transfer buffer to wick.  Take care to eliminate bubbles or air pockets between layers. 
4. Place ice tray in buffer tank and fill 1/2 full with transfer buffer. 
5. Clamp the gel-transfer assembly and insert the assembly in to the electrode assembly.  Immediately insert electrode assembly with transfer assembly in to the buffer tank allowing minimum transfer buffer to drain from the transfer assembly.  Lost transfer buffer in the transfer assembly can cause air pockets which causes spotting or voids in your western development.   Be certain that the all assemblies are appropriately oriented in the buffer tank.
6. Top off buffer tank with transfer buffer and run transfer at a constant current of 70 mA/200 V for 40 – 60 minutes or until the migrating gel line reaches the bottom of the plate.  Conditions of transfer may vary.  Replace melted ice trays as needed during transfer.
7. Remove nitrocellulose blot from clamp assembly and cut the top corner to indicate lane one.
8. Stain blot in 0.01mM amido black to visualize transfer.  Wash with ddH2O.
9. Place blot in blocking buffer (10% casein in PBS) for 4 hours at RT.
10. Wash blot thoroughly (3 X ddH2O, 3 X PBS).
11. Dilute 1° AB with PBS as determined by the principle investigator.  Incubate with agitation for 2-3 hours at RT.
12. Discard 1° AB and wash according to step 10.
13. Dilute 2° AB with PBS as determined by the principle investigator.  Incubate with agitation for 2-3 hours at RT
14. Discard 2° AB and wash according to step 10.
15. If using a biotinylated  2° AB then goto step 16. If using HRP or AP then goto step 18.
16. Dilute streptavidin-enzyme conjugate with PBS as determined by the principle investigator.  Incubate with agitation for 1-2 hours at RT.
17. Discard streptavidin-enz. conjugate according to step 10.
18. Dilute and incubate with HRP or AP according to individual manufacturer requirements.