What can protein extraction buffer change in a Western blot?

The chosen extraction buffer can actually change the experimental outcome quite a lot, as in this example to the right.

Anti-VDE antibodies were used on extracts from Brachypodium distachyon, Arabidopsis thaliana, Oryza sativa.

Adjustment of the buffer to 7M Urea, 2M Thiourea, 4% CHAPS, 35 mM Tris pH 8 resulted in deteciton of VDE in not only Arabidopsis thaliana, but two more species, namely Brachypodium distachyon and Oryza sativa.

If the antibody you are working with is not giving you a signal for more than one species, but the antigen used to elicit this antibody is perfectly conserved in the sequence of the proteins from other species, specific adjustments of the Western blot protocol are necessary.

Usually, lack of detection in additional model species may be connected with epitope inaccessibility. Therefore, application of more denaturing conditions can be helpful to resolve such issue.

Left panel no urea in extraction buffer
Brachypodium distachyon, Arabidopsis thaliana, Oryza sativa

Right panel urea in extraction buffer
Oryza sativa, Brachypodium distachyon, Arabidopsis thaliana

Presented result is a courtesy of Dr. Nelson Saibo,Plant Gene Regulation Lab (GPlantS Unit), ITQB NOVA, Portugal

What can protein extraction buffer change in a Western blot?

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