Increase length and/or number of washing steps (minimum of 5 x 5 min).Insufficient washing between incubation steps Do not use milk to block membranes when using avidin-biotin system because milk contains biotin. Try other blocking reagents, e.g., albumin, gelatin, BSA, casein, or nonfat dry milk (see Bio-Rad Detergents and Blocking Reagents.Add second membrane sheet to bind excess proteinĪntibody binding to proteins in blocking buffer (e.g., phosphospecific antibody binding casein or secondary antibody binding to blocking reagent).Reduce concentration of SDS in transfer buffer.Optimize sample loading see Determining the Appropriate Sample Load for Western Blots Protocol.(both may cause proteins to pass through blot and recirculate in buffer) Make sure all incubation trays are fully cleaned between experiments.Use a dot-blotting trial to optimize antibody concentrations.Reduce/optimize primary and/or secondary antibody concentrations.If using colorimetric reagent, remove the blot from the substrate solution when the signal-to-noise level is acceptable, and immerse in deionized water.Reduce incubation time with detection substrate.Use a pure protein such as BSA or casein as a blocker (see Detergents and Blocking Reagents).See Bio-Rad Detergents and Blocking Reagents Use a different blocking reagent (albumin, gelatin, BSA, casein, or nonfat dry milk).Increase temperature at which blocking is performed, (up to room temp).Increase the duration of the blocking step (overnight at 4☌ instead of 1 hour at room temperature, or a longer incubation at room temperature).Increase the concentration of blocker (e.g., 3–5% BSA, casein, or nonfat dry milk).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.Visualize total protein on gels and blots using Bio-Rad’s Stain-Free Gels featuring our proprietary Stain-Free Technology).To verify protein transfer, stain the membrane with Ponceau S after blotting.To determine if there is residual, untransferred protein remaining on the gel, use a total protein stain on the gel after transfer, e.g., Coomassie, colloidal gold, or SYPRO Ruby.For example, Coomassie and colloidal gold are not compatible with downstream steps (see Bio-Rad Protein Stains and the Protein Stain Selection Guide). If planning to use the blot in downstream steps, make sure that your stain can be removed or is compatible with antibody detection. When using a tank blotter, check the quality of the sponges (foam pads) if a snug assembly can no longer be made between the frames, the pads should be replacedįoam pads for Bio-Rad wet tank blotting systems:Īs a diagnostic test, you can check transfer quality by imaging proteins on the gel and blot.Consider using a rapid transfer pack with the Trans-Blot ® Turbo™ Rapid Blotting System to help prevent improper sandwich assembly.Ensure all filter papers and sponges used in assembly are properly saturated and equilibrated with transfer buffer Use more buffer when assembling transfer sandwich.Carefully remove air bubbles between the gel and the membrane before protein transfer using a roller or glass rod.Improper assembly of transfer sandwich, trapped air bubbles White spots or white areas on the blot may be due to trapped air bubbles or unwetted membrane areas where protein will not bind.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |