Novex™ AP Mouse Chemiluminescent Detection Kit

Here are possible causes and solutions:

- Poor or incomplete transfer: Repeat blot.
- Membrane pads are dirty or contaminated: Soak with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
- Membrane not completely wet: Follow instructions for prewetting the membrane.
- Membrane is contaminated by fingerprints or keratin proteins: Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
- Uneven blocking: The incubation dish must be small enough to allow thorough coverage of membrane.
- Ink used to label membrane: Any labeling of the membrane with ink should be limited to the low MW region of the blot.
- iBind Card damaged: Replace with new card. Ensure that rolling of the membrane on the card is limited to membrane region.
- Membrane is not in proper contact with the iBind Card: Place the membrane on the iBind Card immediately after adding a 1 mL pool of 1X iBind Solution/ iBind FD Solution. Use the roller provided to ensure proper contact.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- iBind Card damaged: Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane&#!48;.
- Stack wet prior to run: Ensure that 5 mL of 1X iBind Solution/ iBind FD Solution is added to the flat region of the iBind Card. Avoid adding solution to the Stack.
- Improper preparation of iBind Solution/ iBind FD Solution: Prepare 1X iBind Solution/ iBind FD Solution as directed in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- Protein is overloaded: Reduce load or dilute concentration of sample.
- Poor or incomplete transfer: Repeat blot.
- Primary antibody is too concentrated: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- Poor or incomplete transfer: Repeat blot. After blotting, stain membrane to measure transfer efficiency. Use positive control and/or molecular weight marker.
- Membrane not completely wet: Follow instructions for prewetting the membrane.
- Primary antibody concentration too low: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
- Inactive primary antibody: Determine activity by performing a dot-blot.
- Low affinity of primary antibody to antigen: Obtain a higher affinity primary antibody.
- Contaminated secondary antibody solution: Wear gloves at all times and keep bottles tightly capped when not in use. Use only purified water when preparing reagents.
- Protein of interest ran off the gel: Match gel separation range to size of protein being transferred.
- Poor retention of proteins: Match gel separation range to size of protein being transferred. Use a molecular weight marker with relevant size proteins. Larger proteins require more transfer time, smaller proteins less. Use membrane with the appropriate binding capacity.
- Improper preparation of iBind Solution/ iBind FD Solution: Prepare 1X iBind Solution/ iBind FD Solution as directed in the manual.
- Improper application of solutions to iBind Wells: Ensure that the solutions are added to the correct wells and that the wells are loaded in numerical order.
- Blot improperly placed on iBind Card: Ensure that the protein side of the blot is in contact with the iBind Card and is placed in the region labeled “membrane”.
- Stack wet prior to run: Ensure that 5 mL of 1X iBind Solution/ iBind FD Solution is added to the flat region of the iBind Card. Avoid adding solution to the Stack.
- Cross-contamination of solutions in wells: Do not move the iBind Western Device during the run.
- iBind Card damaged: Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane”.
- Membrane is not in proper contact with the iBind Card: Place the membrane on the iBind Card immediately after adding a 1 mL pool of 1X iBind Solution/ iBind FD Solution. Use the roller provided to ensure proper contact.
- Device opened prior to completion of run: The device should not be opened once the card has been placed in the device. Re-sealing of the wells on the card can result in leaks.
- 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.
- Protein weakly bound to membrane: Ensure that transfer buffer contains 10-20% methanol.
- Insufficient exposure time: Re-expose film for a longer period of time.
- Insufficient substrate incubation: Perform each step for the specified amount of time or remove blot from substrate when signal-to-noise ratio is acceptable.
- Substrate is contaminated: Wear gloves at all times and keep bottles tightly capped when not in use.
- Blots are too old: Protein may have broken down over time. Use freshly prepared blots.


Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting 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.
- Primary antibody too concentrated: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
- Insufficient removal of SDS/weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection, as directed in the manual.
- Affinity of the primary antibody for the protein standards: Check with protein standard manufacturer for homologies with primary antibody.
- Improper preparation of iBind Solution/ iBind FD Solution: Prepare 1X iBind Solution/ iBind FD Solution as directed in the manual.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.