Invitrogen™ iBind™ Western Starter 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.

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).

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.

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.

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.

Here are possible causes and solutions:

- Membrane not completely wet: Follow instructions for prewetting the membrane. Use an incubation dish which is small enough to allow thorough coverage of membrane to prevent drying out.
Note: If PVDF membrane is being used, we recommend making sure that it is activated with methanol, especially if it has dried up. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into 1X iBind Solution/ iBind FD Solution.
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Film overexposed or became wet during exposure: Decrease exposure time or allow signal to further decay. Prevent leakage by encasing membrane in transparency film and blotting excess substrate from edges before exposure.
- Solutions or incubation tray are contaminated: Use clean glassware and purified water to prepare solutions. Replace or clean the tray thoroughly with a glassware-cleaning detergent. Rinse thoroughly with purified water. Wear clean gloves at all times.
- Concentrated primary antibody used: Follow the instructions provided in the manual ((https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf)) to dilute the primary antibody or determine the optimum concentration by dot blotting.
- Incorrect chemiluminescent substrate used for PVDF: Make sure CDP-Star reagent without enhancer is used.
- Blot is overdeveloped: Follow recommended developing time or remove blot from substrate when signal-to-noise ratio is acceptable.
- Ink used to label membrane: Any labeling of the membrane with ink should be limited to the low MW region of the blot.
- 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: Add the appropriate solutions for each well in the correct numerical order as specified on Page 18 in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf).
- Blot improperly placed on iBind Card: 1) Place the membrane in the designated Membrane Region on the iBind Card. 2) The protein side of the blot should be in contact with the iBind Card. 3) The low MW regions should be closest to the Stack. 4) The membrane should not be in contact with the Stack.

Here are some additional tips to reduce background:

- If iBlot PVDF stacks are used, check that they have not expired as background increases with age. Using the iBlot 2 PVDF stacks will help in reducing the background.
- Make sure that the blot is rinsed in distilled water prior to adding the substrate. Do not rinse in PBS or TBS.

Besides being ~1.7 times as wide, the iBind Flex Card also includes alignment lines printed on the back that appear when solution is added to the card to help in proper horizontal placement of multiple mini or vertically cut strip blots.

The formulations are identical for both the standard and Fluorescent Detection (FD) solutions. The only difference is in the amount supplied, so the iBind Flex Solution Kits are sufficient for 10 midi, 20 mini, or 60 vertically cut strip blots.

Please use the protocol mentioned on Page 13 of the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/ibind_man.pdf) when using the iBind Western System in conjunction with the LI-COR Odyssey Imaging System. We do not recommend using the LI-COR solution.

If the PVDF membrane has dried up, it is important to pre-activate it with 100% methanol and then rinse in distilled water before immersing it in iBind Solution/iBind Fluorescent Detection (FD) Solution. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into iBind Solution/iBind Fluorescent Detection (FD) Solution.

Rinse the iBind Western Device under running water after each use and allow the device to dry before additional usage. No use of detergent is recommended as any residual detergent may interfere with results. To maximize the life of the springs in the iBind Western Device, store the device with latch unlocked, and the lid open as shown on Page 23 of the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf).

It is sufficient to block the membrane in iBind Solution/ iBind Fluorescent Detection (FD) Solution for the time prior to making antibody dilutions. It is not necessary to block the membrane overnight in the blocking solution. The membrane will be subjected to additional blocking in the iBind Solution/ iBind Fluorescent Detection (FD) Solution before primary antibody reaches the membrane.

Yes, membranes that are smaller than 9 cm x 9 cm can be used with the iBind Western device. It is recommended to place the strips or smaller blots centrally. When using larger membranes, care must be taken to ensure that no part of the membrane is in contact with the stack on the iBind card.

We do not recommend using your own solutions. The iBind Solution and iBind Fluorescent Detection (FD) Solution have specific viscosity and are optimized for the sequential lateral flow that is the principle of the iBind Western device. We cannot guarantee the performance with any other solutions.

The original iBind Western System (Cat. No. SLF1000) consists of:
- iBind Western Device
- iBind Blotting Roller
- iBind Window Cover

You would need to purchase the following items separately:
- iBind Cards (Cat. No. SLF1010)
- iBind Solution Kit (Cat. No. SLF1020) for preparing blocking, dilution, and washing buffers for chemiluminescent or chromogenic detection
- iBind Fluorescent Detection Solution Kit (Cat. No. SLF1019) for preparing blocking, dilution, and washing buffers for fluorescent detection
- Invitrogen AP Mouse Chemiluminescent Detection Kit (Cat. No. SLF1021) or Invitrogen AP Rabbit Chemiluminescent Detection Kit (Cat. No. SLF1022)

iBind Western Systems are benchtop devices that automate immunodetection steps. These devices use no external power source, and rely on mechanical pressure to generate sequential lateral flow (SLF) of immunodetection reagents to perform hands-free blocking, antibody binding, and washes for western detection workflows.

Two iBind Western Systems are available:
i) containing the original iBind Western Device, which accommodates one mini blot at a time
ii) containing the iBind Flex Western Device, which accommodates one or two mini blots, one midi blot, or up to six vertically cut membrane strips (for separate antibodies) at a time.