The Western Blot technique for identification of specific proteins may have a number of problems that need to be resolved. One of these is high background staining.
Situation: no or weak signal
Problem
Possible Solution
antibody concentration too high
Decrease the concentration of primary and/or secondary antibody.
incompatible blocking buffer
Do not use milk with an avidin-biotin system. Avoid phosphate-based buffers when probing for phosphoproteins. Test for cross-reactivity in blocking buffer by blocking a clean piece of membrane incubating with antibodies then detecting with substrate of choice. Use Tris-buffered saline for blocking buffer if using an alkaline-phosphatase conjugate. Try a different blocking buffer.
insufficient blocking of nonspecific sites
Increase the concentration of protein in the blocking buffer. Optimize blocking time and/or temperature. Add Tween 80 detergent to the blocking buffer to 0.05% concentration. Prepare antibody dilutions in blocking buffer that contains 0.05% Tween 80 detergent. Use a blot enhancer to reduce background and enhance detection of weakly immunoreactive or low concentration antigens.
insufficient washing
Increase washing (number of washes, volume of buffer). Add Tween 20 detergent to buffer to 0.05%.
membrane handled improperly
Follow manufacturer's instruction to wet and activate membrane. Wear clean gloves or use forceps to handle membrane. Keep the membrane from drying by keeping covered in liquid. Use agitation during all incubations. Handle membrane carefully avoiding damage.
contamination of equipment and materials
Prepare fresh buffers then filter. Make sure that all equipment is clean and contaminant-free.
signal from chemiluminescent substrate too strong
Reduce the length of time that the blot is exposed to film. Reduce the concentration of the substrate. Shorten the incubation time of the membrane with substrate. Completely remove substrate after incubation period. Decrease the concentration of antibodies.
buffers too old
Prepare fresh buffers then filter.
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