WesternDot™ 625 Western Blot Kits

Any dye in the 625 nm emission range should work. For example, you can use the WesternDot 625 Western Blot Kit. We also offer our White-Light Conversion Screen (Cat. No. 4473061), which converts blue light emitted by the blue-light transilluminator or UV light emitted by the UV-light transilluminator to white light. This conversion screen is compatible with multiple protein stains including SimplyBlue SafeStain, SilverQuest silver, and Coomassie blue stains.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are possible causes and solutions:

- Membrane contaminated by fingerprints or keratin proteins: Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
- Concentrated secondary antibody used: Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
- Concentrated Primary antibody used: Decrease the concentration of the primary antibody.
- Affinity of the primary antibody for the protein standards: Check with the protein standard manufacturer for homologies with primary antibody.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are possible causes and solutions for weak/no signal:

- Poor or incomplete transfer: Check transfer conditions, and repeat blot. Use positive control and/or molecular weight marker.
- Nitrocellulose membrane not completely wetted, or PVDF membrane not completely reactivated: Follow instructions for pre-wetting or reactivating the membrane.
- Secondary antibody concentration too low: Use the recommended secondary antibody concentrations.
- Primary antibody concentration too low: Use twice the concentration of primary antibody required for a standard immunodetection. If the signal is still low and the background is not high, increase the concentration.
- Inactive primary antibody: Determine activity by performing a dot-blot or other methods.
- Low affinity of primary antibody to antige: Obtain a higher affinity primary antibody.
- Sample improperly prepared; antigenicity weakened, or destroyed: SDS and reducing agents may interfere with some antibody/antigen affinities.
- Sample too dilute: Load a higher concentration or amount of protein onto the gel.
- Blots are too old: Protein may have broken down over time. Use freshly prepared blots.
- Protein of interest ran off the gel: Match gel separation range to the size of the protein being transferred.
- Poor retention of proteins: Larger proteins require more transfer time, while smaller proteins require less transfer time. Use a molecular weight marker with relevant size proteins. Use membrane with the appropriate binding capacity.
- WesternDot reagents have been frozen: Qdot probes will irreversibly aggregate at freezing temperatures

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are possible causes and solutions:

- Insufficient blocking or non-specific binding: Try a different blocking reagent or increase the concentration of the blocking reagent. We generally obtain good results with 2% casein, 5% non-fat dry milk, or 1/2x fish serum.
- Membrane was blocked with BSA: Do not use BSA-containing solutions for blocking or incubating WesternDot conjugates. For primary antibodies that are incompatible with casein or milk (e.g., many anti-phosphoprotein antibodies), use fish serum or use a 0.5% BSA-containing solution for primary antibody incubation only and then switch to 2% casein or 5% non-fat milk for all other incubation steps.
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Higher intrinsic background with PVDF membranes: Switch to nitrocellulose membranes.
- Nitrocellulose membrane not completely wetted: Follow instructions for pre-wetting the membrane.
- Insufficient washing: Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
- Concentration of primary and/or secondary antibody is too high: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

If there is a large amount of precipitate, it likely indicates that the reagent has been frozen and should be discarded. Qdot probes will irreversibly aggregate at freezing temperatures. The formation of a small amount of precipitate during storage at 2-8 degrees C is normal. We recommend spinning the WesternDot reagent tube down briefly in a microcentrifuge with every use to remove any minor precipitate that has formed during storage and use only the supernatant.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.