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Invitrogen™ iBind™ Flex Fluorescent Detection (FD) Solution Kit

Catalog No. SLF2019
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Catalog No. SLF2019 Supplier Invitrogen™ Supplier No. SLF2019
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Description

Detection kit for use with iBind™ Flex Western System

iBind™ Flex Solution is a proprietary formulation compatible with downstream fluorescent detection/chemiluminescent detection. It serves as a combined blocking, washing and antibody diluent solution to process up to one midi blot, two mini blots, or six vertically cut strip blots when used with the iBind™ Flex Western Device and iBind™ Flex Cards.

Each kit contains reagents sufficient for 10 midi blots or 20 mini blots

Fluorescent Detection (FD) Solution (Contents)

  • iBind Flex FD 5X Buffer
  • iBind Flex 100X Additive
  • iBind Flex FD 10% SDS

Chemiluminescent Solution (Contents)

  • iBind Flex 5X Buffer
  • iBind Flex 100X Additive

Store at 2° to 8°C

Specifications

Product Line iBind
Membrane Compatibility Nitrocellulose, PVDF
Detection Method Fluorescence
Product Type Flex Solution Kit
Shipping Condition Approved for shipment at Room Temperature or on Wet Ice
Content And Storage • iBind Flex FD 5X Buffer
• iBind Flex 100X Additive
• iBind Flex FD 10% SDS

Store at 2–8°C.
No. of Reactions 10 midi blots or 20 mini blots
For Use With (Equipment) iBind Flex Western Device
Kit Contents iBind™ Flex FD 5X buffer, iBind™ Flex 100X additive, iBind™ Flex FD 10% SDS
Quantity 1 kit

Frequently Asked Questions (FAQs)

Can I use conjugated primary antibodies with the iBind Flex Western Device?

Yes. When using a conjugated primary antibody and no secondary antibody, add the diluted primary antibody to well 1 and replace diluted secondary antibody (well 3) with 1X iBind Flex/iBind Flex FD Solution. Alternatively, for faster time to results, add the diluted primary antibody to well 1, leave wells 2 and 3 empty, and add 1X iBind Flex/ iBind Flex FD Solution to well 4. This procedure will save time (~60 min) but may result in higher background.

What procedure should I follow when stripping and reprobing my blot processed using the iBind Western Device or iBind Flex Western Device?

To strip and reprobe your blot processed using the iBind Western Device or iBind Flex Western Device, first strip the blot outside the system using your preferred protocol. After the stripping protocol, wash the blot in distilled water for 5 min. Reblocking is unnecessary as the iBind Solution will perform this step during reprobing. Finally, reprobe with new antibodies using the iBind Western Device or iBind Flex Western Device, starting with the membrane preparation step.

Can I multiplex antibodies using the iBind Western Device or iBind Flex Western Device?

Yes, for each midi blot, mini blot, or vertically cut strip, prepare appropriate primary antibodies together in a single tube of iBind FD/ iBind Flex FD Solution, Prepare appropriate secondary antibodies together in a different tube of iBind FD/ iBind Flex FD Solution. Do not mix primary and secondary antibodies prior to loading in the device. All primary and secondary antibodies should be diluted according to the recommendations outlined in the user manual or quick reference card. When detecting several targets of the same species, the same concentration of secondary antibody can be used as when detecting one target. There is no need to increase the concentration for each additional target. Load multiplexed antibodies into the device as normal.
Caution: Consider cross-reactivity of secondary antibodies when multiplexing (e.g., rabbit anti-goat IgG and goat anti-mouse IgG are likely to cross-react).

Can I use the iBind Western Device or iBind Flex Western Device with Alexa Fluor Plus conjugated secondary antibodies?

Yes, both the iBind Western Device and iBind Flex Western Device work well with Alexa Fluor Plus conjugated secondary antibodies.

With the iBind Western Device and iBind Flex Western Device, how do I know if I am rolling the membrane firmly enough?

When firmly rolling, the iBind/iBind Flex Card will slightly dip in the membrane area, causing the top and bottom of the card to lift. See the iBind/iBind Flex user manual for additional details.
I used the iBind Flex Western system and my membrane is very "spotted". What could have caused this?

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 protein: 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 Flex 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 Flex Card: Place the membrane on the iBind Flex Card immediately after adding a 1 mL pool of 1X iBind Flex Solution/ iBind Flex FD Solution. Use the roller provided to ensure proper contact.
With the iBind Flex Western system, lately, my run time has been in excess of 3 hours. What is the problem?

Here are possible causes and solutions:

- iBind Flex Card damaged: Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane”.
- Stack wet prior to run: Ensure that 5 mL of 1X iBind Flex Solution/ iBind Flex FD Solution is added to the flat region of the iBind Flex Card. Avoid adding solution to the Stack.
- Improper preparation of iBind Flex Solution/ iBind Flex FD Solution: Prepare 1X iBind Flex Solution/ iBind Flex FD Solution as directed in the manual.
I used the iBind Flex Western System and have got a large, scattered signal. What could have caused this?

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.
I used the iBind Flex Western System and am getting a very weak/almost no signal. What is the problem?

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 Flex Solution/ iBind Flex FD Solution: Prepare 1X iBind Flex Solution/ iBind Flex FD Solution as directed in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_flex_man.pdf).
- Improper application of solutions to iBind Flex 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 Flex Card: Ensure that the protein side of the blot is in contact with the iBind Flex Card and is placed in the region labeled “membrane”.
- Stack wet prior to run: Ensure that 5 mL of 1X iBind Flex Solution/ iBind Flex FD Solution is added to the flat region of the iBind Flex Card. Avoid adding solution to the Stack.
- Cross-contamination of solutions in wells: Do not move the iBind Flex Western Device during the run.
- iBind Flex 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 Flex Card: Place the membrane on the iBind Flex Card immediately after adding a 1 mL pool of 1X iBind Flex Solution/ iBind Flex 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.
With the iBind Flex Western System, I am getting a lot of non-specific binding. Can you offer some tips?

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 (https://tools.thermofisher.com/content/sfs/manuals/ibind_flex_man.pdf).
- Affinity of the primary antibody for the protein standards: Check with protein standard manufacturer for homologies with primary antibody.
- Improper preparation of iBind Flex Solution/ iBind Flex FD Solution: Prepare 1X iBind Flex Solution/ iBind Flex FD Solution as directed in the manual.
I used the iBind Flex Western system and am getting high background. What should I do?

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 Flex Solution/ iBind Flex 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 supplier's recommended dilution 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 Flex Solution/ iBind Flex FD Solution: Prepare 1X iBind Flex Solution/ iBind Flex FD Solution as directed in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_flex_man.pdf).
- Improper application of solutions to iBind Flex Wells: Add the appropriate solutions for each well in the correct numerical order as specified in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_flex_man.pdf).
- Blot improperly placed on iBind Flex Card: 1) Place the membrane in the designated Membrane Region on the iBind Flex Card. 2) The protein side of the blot should be in contact with the iBind Flex Card. 3) The low MW regions should be closest to the Stack. 4) The membrane should not be in contact with the Stack.
- Card stack wet prior to run: Ensure that 10 mL of 1X iBind Flex/iBind Flex FD Solution is added to the flow region of the card. Avoid adding the solution to 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.
Is it easy to change the well inserts on the iBind Flex Western Device?

Yes. Due to an intuitive magnetic design, it is very simple for the user to change the well inserts. However, we highly recommend the user handle the well inserts with care (as engraved on each insert) when changing or washing, and store the other two well inserts in the convenient underside tray when not in use.
Is there any difference between the iBind Flex Card and the original iBind Card?

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.
Is there any difference between the iBind Flex Solution Kits and the original iBind Solution Kits?

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.
Is the iBind Flex Western Device compatible with horizontally cut strip blots that are rotated 90 degrees and processed vertically?

It is not recommended due to the potential for non-uniform flow from top to bottom which could result in a gradient signal intensity across horizontally cut strip blots.
Does the original iBind Card fit on the iBind Flex Western Device?

Yes, to run a single mini blot or up to three vertically cut strip blots. However, when using the Mini or Multi-Strip Inserts, wells not in contact with the card should remain empty.
Can you run only one mini blot or less than six vertically cut strip blots on the iBind Flex Western Device when using the iBind Flex Card?

Yes. Whether using the Mini or Multi-Strip Inserts, empty wells are still in contact with the card and should be filled with water to ensure proper flow of all solutions across the card.
For the iBind Flex Western System, do you have a protocol for detecting the signal using the LI-COR Odyssey Imaging System?

Please use the protocol mentioned on Page 15 of the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/ibind_flex_man.pdf) when using the iBind Flex Western System in conjunction with the LI-COR Odyssey Imaging System. We do not recommend using the LI-COR solution.
What iBind Flex Solution kits are available?

We offer the iBind Flex Solution kit and the iBind Flex Fluorescent Detection (FD) Solution kit. For standard chemiluminescent or chromogenic detection, we recommend the iBind Flex Solution kit (Cat. Nos. SLF2020, SLF2020X4) and for fluorescent detection, we recommend the iBind Flex Fluorescent Detection (FD) Solution kit (Cat. No. SLF2019).
When is it necessary to pre-activate a PVDF membrane with methanol for use in the iBind Flex Western Device?

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 Flex Solution/iBind Flex 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 Flex Solution/iBind Flex Fluorescent Detection (FD) Solution.
What are your recommendations for cleaning and maintenance of the iBind Flex Western device?

- Rinse the iBind Flex well inserts under running water after each use and allow the well inserts to dry before additional usage.
- Handle well inserts with care.
- Store unused well inserts in the drawer in the iBind Flex Western Device.
- To maximize the life of the springs in iBind Flex Western Device, store the device with latch unlocked, and the lid open as shown on Page 28 of the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_flex_man.pdf).
What is the recommended amount of time to immerse the membrane in iBind Flex Solution/iBind Flex Fluorescent Detection (FD) Solution prior to assembly in the iBind Flex Western device?

It is sufficient to block the membrane in iBind Flex Solution/ iBind Flex 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 Flex Fluorescent Detection (FD) Solution before primary antibody reaches the membrane.
Can I use my own blocking reagent or antibody incubation solution with the iBind Flex Western System?

We do not recommend using your own solutions. The iBind Flex Solution and iBind Flex Fluorescent Detection (FD) Solution have specific viscosity and are optimized for the sequential lateral flow that is the principle of the iBind Flex Western device. We cannot guarantee the performance with any other solutions.
What are the components of the iBind Flex Western System and which components do I need to purchase separately?

The iBind Flex Western System (Cat. No. SLF2000) consists of:
- iBind Flex Western Device
- iBind Flex Midi Insert
- iBind Flex Mini Insert
- iBind Flex Multi-Strip Insert
- iBind Flex Blotting Roller

You would need to purchase the following items separately:
- iBind Flex Cards (Cat. No. SLF2010)
- iBind Flex Solution Kit (Cat. No. SLF2020) for preparing blocking, dilution, and washing buffers for chemiluminescent or chromogenic detection
- iBind Flex Fluorescent Detection Solution Kit (Cat. No. SLF2019) 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)
How do iBind Western Systems work?

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.

For Research Use Only. Not for use in diagnostic procedures.