Optimize transfer protocol for specific protein either by increasing the transfer time or increasing the voltage.Optimize the concentration of the blocker.Try using ECL western blot rather than the colorimetric western blot.Concentrate your protein lysates by isolating the cellular compartment containing your protein of interest (ie.Use an enrichment step to maximize the signal.The protein of interest in not abundantly present in the tissue: It also may be necessary to induce cells before harvest.Load sufficient protein onto the gel (~20-30 µg).Unfortunately this might require a new vial to be used instead. Antibody was not stored as recommended.It is best to create aliquots of smaller amounts as soon as the product arrives at your location. Freeze/thaw cycles are detrimental and can cause degradation.Biotinylated antibodies should not be used with milk or casein.HRP should not be used in conjunction with sodium azide (1% or greater) or hemoglobin.Mouse Anti-HSP70) use an anti-mouse secondary (ie. For example, if the primary was raised in mouse (ie. The secondary antibody should be raised against the host species of the primary antibody. Primary and secondary antibodies are incompatible: Use a positive control with all experiments. Check the datasheet/reference materials or perform a BLAST alignment to see whether the antibody should react with the target protein.Primary antibody does not recognize the antigen: Always optimize both primary and secondary antibodies with every experiment.Too high- this is indicated with ghost/hollow bands.Too low- use a higher concentration of antibody, or try incubating longer.Commercialization/Collaboration Programįollow our western blot troubleshooting guide to quickly target the potential cause of a problem with your western blot protocol, and test out solutions.įirst identify the problem with your western blot from the options below: Weak or No Signal.Neurodegenerative Protein Handling Instructions.Proteins for Neurodegenerative Disease Research.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |