Bio-Rad Trans-Blot® Cell - Manual (Page 23)

3. Activity test for the first antibody solution.
• Use an ELISA, RID, Ouchterlony immunodiffusion, or precipitation test
to determine reactivity of the antibody with the antigen. If possible,
repeat the assay procedure with several dilutions of first antibody solu-
tion.
Total Protein Detection
Colloidal Gold Total Protein Stain — High Background
1. The blocking step is insufficient or omitted.
• Block with 0.3% Tween-20 in TBS, using three washes of 20 minutes
each.
2. The membrane used is not compatible with this stain.
• Positively charged nylons cannot be used with Colloidal Gold stain. Use
the Biotin-Blot Total Protein Detection Kit instead.
3. Contamination of the membrane occurred at a previous step, i.e. elec-
trophoresis or transfer.
• Discard and remake the gel and transfer solutions.
• Replace or thoroughly clean contaminated fiber pads.
4. Excessive amounts of protein are loaded on the gel, or too much SDS is
used in the transfer buffer. Proteins can pass through the membrane with-
out binding and recirculate through a tank blotting system
• Reduce the amount of protein on the gel or SDS in the transfer buffer.
Add a second sheet of membrane to bind excess protein.
5. Colloidal gold stain solution is contaminated.
• The stain is a reusable reagent. Be sure to use a separate, clean plastic
container to store previously used reagent in the refrigerator. Discard
any reagent that has viscous sediment at the bottom of the bottle. If the
solution does not have a dark burgundy color, but is a light blue, the
stain was contaminated with buffer salts. Buffer salts will react with the
gold sol causing non-specific precipitation of the reagent onto the mem-
brane. Discard this solution.
Colloidal Gold Total Protein Stain — Low Sensitivity
1. Increase the incubation time for detection of low level signals.
• Overnight incubations are possible, although background staining can
increase.
2. Transfer is incomplete.
• See poor transfer for suggestions on how to enhance transfer efficiency.
3. Stain is exhausted, as evidenced by the loss of the dark burgundy color and
longer staining times.
• Discard the reagent.
4. Buffer salt contamination has occurred. The solution will be light blue
instead of dark burgundy.
• Discard the reagent.
5. The sample load may be too low for the reagent to detect.
• Use the Gold Enhancement Kit for detection levels as low as 10 pg of
protein per band.
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