Too low a concentration will also result in a weak or no signal. An indication of this would be brown/yellow bands on the membrane or ghost/ hollow bands on the films. Too high a concentration of antibody can not only cause high backgrounds, but can also use up all the substrate before it can be detected, resulting in no or weak signal. If possible use higher levels of antigen. Low levels of antigen on the membrane, as a result of poor transfer or insufficient loaded on gel, may be a cause. For small molecular weight proteins reduce transfer times and use membranes with smaller pore sizes. The use of 20% methanol in transfer buffers should improve transfer. If after checking the membrane and poor transfer has occurred, explore other options such as a unique product from G-Biosciences that reduces transfer times, prevents overheating and aids high molecular weight protein transfer. SWIFT Membrane Stain is a 30 second reversible stain that offers greater user friendly attributes compared to Ponceau-S. A quick quality control step can be performed after transfer to check transfer by using a reversible stain, such as Ponceau-S or commercially available stains.

The presence of trapped air bubbles will prevent protein transfer resulting in blank areas on the developed blot. If there's no protein, there will be no signal. Poor protein transfer to the membrane is a routine, but often overlooked problem. Weak or No Signal Improper Transfer or Protein High Concentration of Antibody Interference from Incompatible Blocking Agent Non-Specific Sites Insufficiently Blocked Insufficient Washing Overexposure of Membrane Poor Handling of Membrane Buffers Contaminated 3. Small gel pieces on the membrane may also cause this effect. Ensure all equipment is clean prior to use. The presence of contaminants on dirty equipment may result in blotchy or speckled patterns. Filter the conjugate through a 0.2µm filter may resolve the issue. Blotchy or Speckled Backgrounds Conjugated Enzyme Antibody ConjugatedĬonjugation of the secondary antibody may result in intense spots on the membrane, producing a speckled pattern. The presence of bacteria or other contaminants will severely affect blotting. Old membranes can also severely affect the quality of your blots Buffers Contaminated If the membrane dries out during the procedure a high background may occur. PVDF requires methanol for proper hydration. Membranes poorly hydrated before transfer of Western blot development. Simple solution: Reduce exposure times! Poor Handling of Membrane For improved washing, use wash buffers with Tween 20. Increase both the volume, length and number of wash steps. Improper washing will result in excess antibody remaining on the blot. The inclusion of Tween 20 at a final concentration of 0.05% can also improve blocking. Factors to investigate are blocker tube (see above), length of incubation and temperature of incubation. Optimization of the blocking conditions is essential for clear backgrounds. Non-Specific Sites Insufficiently Blocked Some blocking agents may actually bind to the antigen of interest and therefore prevent antibody binding and subsequent detection. Other alternatives are protein-free blockers or non mammalian protein blockers.

The best solution is to find a non animal blocking agents, such as G-Biosciences NAP-BLOCKER. As the blocking agent is on the entire surface of the membrane then a uniform signal will be seen. Antibodies produced in animals have an inherent problem in the fact that they can interact with animal proteins used in blocking agents. Many blocking agents are protein based, and some are sera protein based, such as Bovine serum albumin. Interference from Incompatible Blocking Agent High antibody concentration may also result in the development of non-specific bands. High concentrations of antibodies will lead to a coating of the membrane with antibodies due to saturation, resulting in a uniform signal across the entire membrane. Uniform High Background High Concentration of Antibody

Ghost/ Hollow Bands or Brown/ Yellow Bands on Membraneġ.

The aim of this blog is to highlight some of the routine problems that occur with Western blotting and suggest possible solutions.

Transfer western blot