Bio-Rad Trans-Blot® Cell - Manual (Page 24)

Biotin-Blot Total Protein Detection — High Background
1. Blocking conditions are insufficient.
• Match the blocker to the membrane. Nylon membranes require the
addition of 1-methyl-2-pyrrolidinone (MPO) to several solutions.
Consult the Biotin-Blot manual for specific details.
2. Membrane is left in color development solution too long.
• Remove the membrane from the color development solution when the sig-
nal is apparent and the background has not developed. Transfer the blot to
distilled water immediately to stop the development.
3. 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.
Biotin-Blot Total Protein Detection — No Reaction
or Weak Color Development
1. Transfer is incomplete.
• See Poor Transfer for suggestions on how to enhance transfer efficiency.
2. The sample load may be too low for the reagents to detect.
• Increase the amount of protein loaded on the gel.
3. NHS-biotin solution is inactivated.
• NHS-biotin hydrolyzes in aqueous solutions. Equilibrate the reagent vial
to room temperature before opening to prevent condensation of water
inside the container. Use a sterile syringe to remove reagent to prevent
contamination.
• Add the NHS-biotin reagent to the Borate-Tween solution just prior to
use.
4. Amine containing buffer salts compete for the biotinylation reagents.
• Wash the membrane thoroughly in Borate-Tween to remove any residu-
al buffer salts from electrophoresis and transfer.
5. Avidin-HRP conjugate is inactive.
• Follow the activity test procedures to determine if the reagent is inactive.
6. Color development solution is inactive.
• Follow the activity test procedures to determine if the reagent is inactive.
Anionic Dyes — High Background
1. Destaining is insufficient.
• Increase the number and duration of washes with the destaining solu-
tion.
2. Dye solution is too concentrated.
• Remake the solution.
3. Nylon membranes are not compatible with anionic dyes.
• Use the Biotin-Blot Protein Detection Kit.
Anionic Dyes — Low Sensitivity
1. Anionic dye stains do not detect protein bands below ~100 ng.
• Use a more sensitive stain such as the Colloidal Gold stain or the Biotin-
Blot Protein Detection Kit.
• Increase the sample load to achieve the detection level of the anionic dye
stains.
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