Unpurified antibodies can produce non-specific bands: Refer back to the product datasheet/ product page for recommended starting dilutions.Decrease the concentration of the primary antibody, and run a secondary antibody control.Use freshly prepared sample lysates as they will have less degradation and therefore less non-specific bands.PVDF membranes tend to have a higher background. We recommend using a nitrocellulose membrane with a pore size of 0.2 µm.Ensure membranes are thoroughly covered in buffer at all times.Reduce film exposure times to less than 30 seconds.Milk contains casein which is a phospho-protein. For phospho-specific antibodies, always use BSA rather than milk. Add a mild detergent (Tween 20) to the incubation and washing buffer.Always use clean equipment, fresh solutions and wear gloves.Ĭross-reaction between the blocking agent and the primary and secondary antibodies: Add or increase concentration of detergent in wash buffer (ie.Increase the volume and number of washes.We recommend 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.Try a different blocker such as BSA, casein, or milk.Increase blocker incubation time and/or temperature.Reduce the concentration of primary and/or secondary antibodies.Decrease the number of washing steps and/or the duration of washes.Ensure that the secondary antibody tag has not been exposed to excessive light.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.Make sure that the methanol concentration in the transfer buffer is not more than 10–20% and that high-quality, analytical grade methanol is used. It may cause a reduction in pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral. Methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane, but has some negative effects on the gel itself, leading to a decrease in transfer efficiency.We recommend pre-equilibrating the gel in 2x Transfer buffer (without methanol) containing 0.02–0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1x transfer buffer containing 10% methanol and 0.01%SDS. For proteins that are difficult to elute from the gel such as large molecular weight proteins, a small amount of SDS may be added to the transfer buffer to improve transfer. This inhibition is higher for nitrocellulose than for PVDF. SDS in the gel and in the SDS-protein complexes promotes elution of the protein from the gels but inhibits binding of the protein to membranes.Increase voltage, current or length of time for transfer.
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