![]() Careful attention to your handling and protocol steps is required, and multiple trials may be necessary to resolve this problem. If you observe high background across the blot, there are a number of likely causes. See Blotting Membranes and Papers to learn more about the different types available and their properties If the membrane dries, rewet in methanol and re-equilibrate in TBS-T (caution: may adversely affect downstream detection processes) Once wet, do not allow membrane to dry out. Because of the hydrophobic nature of PVDF, the membrane must be prewetted in methanol prior to equilibration in aqueous transfer buffer. PVDF: White regions on PVDF membrane indicate areas where the membrane was either inappropriately prewetted or allowed to dry out.Equilibrate in transfer buffer until ready to use If wetting does not occur immediately by immersion of the sheet in transfer buffer, heat distilled water until just under boiling point and soak the membrane until completely wet. Nitrocellulose: White regions on the nitrocellulose membrane indicate dry areas where protein will not bind.Use a cooling coil or “blue ice” insert in the cellĬooling for Bio-Rad Wet Tank Blotting Systems:ĭried out or improperly hydrated membrane.Perform transfer in tank apparatus placed in ice or a temperature-controlled (4☌) room.Reduce current, and increase transfer time to compensate.High-intensity transfers in tank blotting sample can cause buffer heating, leading to bubble formation ensure that buffer remains cool. ![]()
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