A possible protocol for western blotting troubleshooting? - (Jun/14/2012 )
Hi, my lab has recently encountered a rough patch with regards to western blotting.
To troubleshoot it i was going to suggest the dabbing onto strips on nitrocellulose either: protein rich lysates, primary antibody, or secondary antibody. then block using 5% milk and then do the western blotting protocol from various points
(i.e incubate primary antibody in milk over strip with lysates directly on the nitrocellulose, secodary antibody on the strip with primary antibody directly on it etc.etc.)
Then test the detection reagents with the strips, thefore we will be able to detect at what stage our westerns fail.
A good plan, or is there something more convential to this unconvential crisis?
This is commonly called a dot- or slot- blot. There are heaps of protocols out there for doing them,.