Protein Protocols Protein Protocols

400 Fowler
Table 1
Troubleshooting Immunogold Silver Staining
Observation Probable cause Remedy
Precipitation of silver Glassware: chromic acid was Rinse glassware several times
enhancement mixture before used for rinsing and it was not with 0.1 M HCl and distilled
indicated time interval washed away with HCl. water
Glassware: Traces of metals Avoid contact with metallic
originating from metal parts, objects; do not use glassware
e.g., cleaning brushes, or brush with metallic handle to
originating from previous clean glassware; use disposable
experiments in which metallic plastics instead.
compounds were used, e.g.,
silver staining.
High background and non Microprecipitates that are Incubate for a shorter time in
specific staining macroscopically invisible silver-enhancement mixture.
produce a high background
when the stability time limit is
reached.
Primary antibody is too Optimize dilution of primary
concentrated. antibody using dot-blot assay.
Wrong type of gelatin Use the type of gelatin
gelatin used during incubation prescribed.
with AuroProbe
No staining Difficulties regarding the Perform a dot-blot assay
reactivity of the primary (see Subheading 4.3.)
antibody with the immunogold
reagents
Inefficient transfer from gel to Optimize blotting conditions;
membrane silver-stain gel to see what
remains.
Error in handling: the steps Repeat the procedure in the
were not performed in the right right order.
order or a step was omitted.
Excessive dilution of primary Optimize primary antibody
antibody dilution using a dot-blot assay.
Nonreactive primary antibody Use a primary antibody of
or a primary antibody that was highest possible antibody
destroyed by inappropriate quality, and repeat the
storage conditions procedure with a new batch of
primary antibody.
The gold probe may have been Repeat procedure with fresh
denatured owing to wrong gold probe.
storage conditions.
Signal too weak Excessive dilution of primary Optimize dilution of primary
antibody antibody using a dot-blot assay.
Excessive dilution of Use AuroProbe reagent as
AuroProbe reagent or too short recommended in Subheading 3.2.
an incubation time
Silver-enhancement time Rinse the membrane in distilled
too short water and repeat the silverenhancement
in fresh reagent.