Increase the concentration of the blocker from 5% to 7%.Potential Cause: Nonspecific sites were not blocked Switch to an affinity purified product instead.Potential Cause: Non-specific bands can be produced by unpurified antibodies Try loading less protein into each well.Check the product datasheet/product page for recommendations on starting dilutions.Try decreasing the concentration of the primary antibody then try running a secondary antibody control.Potential Cause: Antibody concentration is too high Make sure that membranes are adequately covered in buffer at all times.Potential Cause: Overexposure of Membrane Milk contains casein which is a phospho-protein. For phospho-specific antibodies, BSA should be used rather than milk. Try adding a mild detergent (Tween 20) to the incubation and washing buffer.Potential Cause: Detection of the blocker by the primary/secondary antibodies: Clean equipment, fresh solutions and gloves should always be used. Potential contaminants should be kept in mind.Increase the concentration of detergent in wash buffer (i.e.Increase the volume and amount of washes.The recommendation is 5% w/v non-fat dry milk in Tris buffered saline with 0.05% TritonX100, TBST overnight at 4 oC or 1 hour at RT.Use a different blocker such as BSA, casein, or milk.Try increasing the concentration of blocker.Try increasing the blocker incubation time and/or the temperature.Both the primary and secondary antibodies should be optimized with every experiment.Try reducing the concentration of primary and/or secondary antibody.Potential Cause: High Antibody Concentration Make sure that the secondary antibody tag was not exposed to excessive light.Potential Cause: Secondary antibody tag not visible Optimize the transfer protocol for the specific protein.Potential Cause: Protein did not transfer Try optimizing the concentration of the blocker.Potential Cause: Epitope is masked by the blocker Try to use ECL western blot as opposed to the colorimetric western blot.mitochondria or cellular membrane) in order to concentrate the protein lysates. Isolate the cellular compartment containing the protein of interest (i.e.Potential Cause: Protein is not highly expressed in the cells/tissue Make sure that the protein is not degraded.It is sometimes necessary to induce cells in order to produce more protein before harvest.Ensure that sufficient protein is loaded onto the gel (~20 µg).Potential Cause: Antigen level is too low If the antibody was not stored in line with recommendations, then unfortunately a new vial might need to be used instead.Aliquot antibodies into one time use vials upon delivery. The antibody structure can degrade with freeze/thaw cycles.Mouse Anti-HSP70) an anti-mouse should be used for the secondary (i.e. For example, if the primary was raised in mouse (i.e. Raise the secondary antibody against the host species of the primary antibody.Potential Cause: Primary and secondary antibody mismatch
Verify that the antibody is definitely able to detect the antigen in the species by checking data sheets and reference material.Potential Cause: Primary antibody does not recognize the antigen Both primary and secondary antibodies should be optimized with every experiment.Use a higher concentration of antibody, or a longer incubation.Potential Cause: Incorrect antibody concentration This western blot troubleshooting guide is designed to help target the potential cause and test out solutions. Jan 28 2020Īt some point in time most people will have difficulty in getting a western blot protocol to work. Sponsored Content by StressMarq Biosciences Inc.
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