Is an antibody not specific or Western blot protocol is not optimized?

Antibody: TROL | Thylakoid rhodanese-like protein, AS19 4257.

Western blot protocol before optimization
Protein load/well: 20 µg
Blocking: 5 % milk
Primary antibody: 1: 2000 ON/4ºC
Secondary antibody: 1: 50 000 1h/RT

Western blot protocol after optimization
Protein load/well: 2 µg
Blocking: 5 % milk
Primary antibody: 1: 2000 ON/4ºC
Secondary antibody: 1: 50 000 1h/RT

Visible improvement was achieved with decreased protein load/well (10x).

Blot image, courtesy of Dr. Lea Vojta and Dr. Hrvoje Fulgosi, Laboratory for Molecular Plant Biology and Biotechnology, Division of Molecular Biology, Institute Ruđer Bošković, Croatia.

In a need to optimize your western blot protocol?
Request a copy of the Agrisera Western blot trouble shooting brochure, or send your inquiry directly to [email protected].		 
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Technical blog

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