Protein Protocols Protein Protocols

402 Fowler
Table 3
Troubleshooting General Protein Staining with AuroDye Forte
Observation Probable cause Remedy
AuroDye forte turns purplish Agglutination of gold particles Wash blot briefly in excess
during staining by proteins released from the distilled water; replace
blot AuroDye forte; if possible,
use lower protein loads; this
observed with PVDF
membrane because it retains
proteins better than
nitrocellulose.
AuroDye forte turns colorless Adsorption of all the gold Replace AuroDye forte and
during staining. particles by excess protein on double the volume used per
the blot cm 2 of blot; if possible use
lower protein loads.
Spotty background Impurities released from filter Use high-quality filter paper
paper adsorbed onto blot during during electroblotting
transfer procedure.
High background Interference by proteinaceous Use extra transfer membrane
contaminants on cathodic side of the gel;
use clean Scotch-Brite pads.
Optional silver-enhancement Use a shorter silvertime
was too long enhancement time.
Interference by chemical Always use high-quality
contaminants chemicals.
Smears on background Incorrect handling Handle blots by their edges
using clean plastic forceps;
avoid contact with gloves.
12. If large amounts of protein are loaded on the gel, when transferred to the membrane they
will not only be heavily stained, but will leak off the membrane from saturated sites.
Excessive protein leakage will cause an aggregation of the gold particles and destroy the
AuroDye forte reagent. The problem is generally more severe with 1-D, than with 2-D
gels, in which the proteins are more spread out. For 1-D gels, it is recommended that
protein loads should be equivalent to amounts capable of giving resolvable bands after
silver staining. Single bands in 1-D gels should not exceed 1000 ng of protein. Molecular
weight standards should be loaded at approx 200 ng/band. In general, the use of lower
protein loadings will give better separation, and samples will be conserved.
13. When staining 2-D gels, they should be thoroughly washed in several changes of excess
transfer buffer after electrophoresis to remove any remaining ampholytes.
14. AuroDye forte is a stabilized gold colloid sol, adjusted to a pH of approx 3. At this low
pH, the negatively charged gold particles bind very selectively to proteins by hydrophobic
and ionic interactions. This may result in different staining intensities, depending on the
isoelectric point of the proteins being stained. However, this has been reported to be a
feature of other protein staining methods, such as silver staining and Coomassie Blue (21).