I have recently started doing western blots in my lab. After transferring my gel onto the nitrocellulose membrane. I wash the blot 1 time with TBST then block with 5% non-fat milk for 1 hour at room temp. I don't wash the blot after blocking. I pour off 5% milk and add 1:1000 dilution of primary antibody in 1% milk at 4 degree overnight. I wash the blot 3 times with TBST in 5 minute intervals then I add the secondary antibody 1:5000 and 1:2000 dilution in 1% milk for one hour. Wash the secondary off with TBST 1 time for 5 minutes then I view on the imager.

Not sure what caused this smear? Any critiques to my protocol would be helpful.