Components

Western Blotting Kit I RD (PBS/PVDF), PN 926-32081

• IRDye® 800CW Goat anti-Mouse Secondary Antibody (0.1 mg)
• IRDye 680RD Goat anti-Rabbit Secondary Antibody (0.1 mg)
• Intercept® (PBS) Blocking Buffer (500 mL)
• Millipore Immobilon®-FL PVDF Membrane (0.45 µm, 10 x 10 cm)

Other Required Reagents

• Primary antibodies

  When using an Western Blotting Kit, the primary antibodies must be from Rabbit or Mouse host species.

• Tween® 20

• TBS or PBS buffer

• Methanol (when using Western Blotting Kit V or VI)

• SDS (when using Western Blotting Kit V or VI)

• Western Blot Incubation Box (appropriate for blot size)
  • Small (7.2 x 4.9 x 3.0 cm) (LI‑COR, PN 929-97101, 929-97105, 929-97110)

  • Medium (8.9 x 6.5 x 2.8 cm) (LI‑COR, PN 929-97201, 929-97205, 929-97210)

  • Large (11.5 x 8.8 x 2.8 cm (LI‑COR, PN 929-97301, 929-97305, 929-97310)

  • X-Large (15.2 x 10.1 x 3.1 cm) (LI‑COR, PN 929-97401, 929-97405, 929-97410)

• Odyssey Pen (LI‑COR, PN 926-71804) (optional)

Western Blot Detection Protocol

Prepare your Western blot using standard blotting procedures and the membrane provided in your Western Blotting Kit. After transfer, allow the blot to dry for an hour before beginning the detection protocol. Dry blots can be stored between pieces of filter paper overnight at room temperature.

Only handle membranes by the edges with clean forceps. Be careful not to touch the membrane with your hands or gloves.

Do not write on membranes with regular ink pens or markers, because the ink will fluoresce on Odyssey Imaging Systems. Use only a pencil to write on PVDF membranes, because the ink from the Odyssey Pen will dissolve in the methanol used to wet the PVDF membrane. You can write on nitrocellulose membranes with pencil or the Odyssey Pen (PN 926-71804).

Incubation Box Volumes

Use the following volumes for blocking and washing steps.

• 5 mL for a Small Box.

• 10 mL for a Medium Box.

• 15 mL for a Large Box.

• 20 mL for an X-Large Box.

Step 1. Wet Membrane

For Immobilon®-FL PVDF Membranes

1. Wet for 30 seconds in 100% methanol.
2. Wet in 1X TBS or PBS for 5 minutes (using the appropriate buffer system).

Step 2. Block the Membrane

Place the membrane in an incubation box and block the membrane with Intercept Blocking Buffer for 1 hour with gentle shaking.

See the Incubation Box Volumes section for recommended volumes.

Step 3. Dilute Primary Antibody

1. Prepare the primary antibody diluent: Add Tween® 20 to Intercept Blocking Buffer for a final concentration of 0.2% Tween 20.

2. Dilute primary antibody in antibody diluent using the vendor's recommendations (see the Incubation Box Volumes section for recommended volumes).

   Depending on the primary antibody, dilutions may range from 1:200 – 1:5,000.

Step 4. Incubate Blot in Diluted Primary Antibody

Incubate the blot for 1 - 4 hours at room temperature or overnight at 4 °C with gentle shaking.

Optimal incubation times vary for different primary antibodies.

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If the procedure cannot be completed in full, this is a good place to stop until the following day. Incubate the primary antibody overnight at 4 °C with gentle shaking.

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Step 5. Wash Membrane

1. Carefully pour off primary antibody solution.
2. Rinse the membrane with 1X TBS or PBS -T (0.1% Tween 20).
3. Cover blot with 1X TBS or PBS-T (see the Incubation Box Volumes section for recommended volumes).
4. Shake vigorously on platform shaker at room temperature for 5 minutes.
5. Pour off wash solution.
6. Repeat 3 additional times.

Step 6. Reconstitute Secondary Antibody

1. Combine each 0.1 mg of secondary antibody with 0.1 mL of sterile distilled water.

2. Gently mix by inverting.

3. Allow the solution to stand at room temperature for at least 30 minutes before use. Protect the solution from light.

   If the solution is not completely dissolved after standing at room temperature, centrifuge the solution.

When stored as recommended, this product is stable for 3 months.

Step 7. Dilute Secondary Antibody

Dilute secondary antibody in the appropriate diluent for the membrane you're using. For IRDye® 800CW secondary antibodies, IRDye® 680RD secondary antibodies, or IRDye® 680LT secondary antibodies, the recommended starting dilution is 1:20,000. For VRDye secondary antibodies, the recommended starting dilution is 1:10,000. The following includes recommendations on antibody dilution buffers.

Secondary Antibody Diluent for Immobilon®-FL PVDF

Use Intercept T20 Antibody Diluent and SDS. Alternatively, add Tween 20 to a final concentration of 0.2% and SDS to a final concentration of 0.01 - 0.02% in Intercept Blocking Buffer.

Step 8. Incubate Blot in Secondary Antibody

Protect membrane from light during incubation.

• Incubate blot in diluted secondary antibody for 1 hour at room temperature with gentle shaking.
• Do not incubate for longer than 1 hour, because the background may increase.

Step 9. Wash Membrane

Protect membrane from light during washes.

1. Carefully pour off secondary antibody solution.
2. Rinse the membrane with 1X TBS or PBS-T (0.1% Tween 20).
3. Cover blot with 1X TBS or PBS-T (see the Incubation Box Volumes section for recommended volumes).
4. Shake vigorously on platform shaker at room temperature for 5 minutes.
5. Pour off wash solution.
6. Repeat 3 additional times.

Step 10. Rinse Membrane

Rinse the membrane with 1X TBS or PBS to remove residual Tween 20.

• Membranes can be stored at 4 °C in TBS or PBS for short periods. Always protect membranes from light.

• Membranes can be stored dry at room temperature for prolonged storage. Always protect membranes from light.

• If you plan to strip and reprobe the Western blot, do not allow the completed Western blot to dry. The stripping process is less effective on Western blots that have been allowed to dry.

Step 11. Scan Membrane

Protect the membrane from light prior to scanning.

Scan the membrane on an Odyssey Imager.

The membrane can be scanned wet or dry. Scanning the membrane dry can add signal intensity, but can also lead to increased background.

Optimization

• Follow the protocol carefully.

• No single blocking buffer will be optimal for every antigen-antibody pair.

  Some primary antibodies may exhibit greatly reduced signal or non-specific binding in different blocking solutions. If you have difficulty detecting your target protein, changing the blocking solution may dramatically improve performance. If the primary antibody has worked well in the past using chemiluminescent detection, try that same blocking solution for near-infrared fluorescent detection on an Odyssey Imaging System.

• To avoid background speckles on blots, use high-quality ultra pure water for buffers. Rinsing previously-used incubation boxes with methanol can reduce background contamination of future blots.

• Always use a consistent buffering system for wash buffers and blocking buffers. (e.g. Intercept (TBS) Blocking Buffer, use TBS as the base for wash buffers; for Intercept (PBS) Blocking Buffer, use PBS as the base for wash buffers).

• Never perform Western incubations or washes in dishes that have been used for Coomassie staining.

• Only handle membranes by the edges with clean forceps. Be careful not to touch the membrane with your hands or gloves.

  After handling membranes that have been incubating in antibody solutions, clean forceps thoroughly with methanol, then rinse with distilled water.

• Always pour off antibody solution and washes from the same corner of the box to ensure complete removal of previous solutions.

• Do not wrap the membrane in plastic when scanning.

Guidelines for Multiplexed Detection

Multiple antigens can be detected simultaneously on the same blot using IRDye® secondary antibodies and VRDye™ secondary antibodies. When performing a blot, use the standard Western blot protocol with the following modifications:

• Combine the primary antibodies in the antibody diluent. Incubate simultaneously with the membrane. The primary antibodies must be from different host species or subclasses if the targets are close in molecular weight.

• Combine the IRDye secondary antibodies and VRDye secondary antibodies in the antibody diluent.

• Incubate simultaneously with the membrane.

Multiplexed detection requires careful selection of primary and secondary antibodies. A comprehensive course is available on the Lambda U® Western Blot Education portal (lambdau.net) to help you select appropriate antibodies. The following guidelines provide information that will help you successfully design multiplexed experiments:

• The primary antibodies must be derived from different host species so that they can be discriminated by secondary antibodies of different specificities (for example, primaries from rabbit and mouse will be discriminated by anti-rabbit and anti-mouse secondary antibodies, respectively).

• If the two primary antibodies are mouse monoclonals from different IgG subclasses (e.g., IgG₁, IgG_2a, or IgG_2b), IRDye® Subclass-Specific secondary antibodies can be used for multiplex detection. The same subclasses cannot be combined in a two-color Western blot (e.g., two IgG₁ primary antibodies).

  See Western Blot and In-Cell Western™ Assay Detection Using IRDye Subclass-Specific Antibodies (licorbio.com/subclass) for more information.

• Anti-goat secondary antibodies cannot be multiplexed with goat-derived secondary antibodies (e.g., donkey anti-goat and goat anti-rabbit). The secondary antibodies will cross-react.

• Before combining primary antibodies in a multiplexed experiment, always perform preliminary blots with each primary antibody alone to determine the expected banding pattern and possible background bands. Slight cross-reactivity may occur and can complicate interpretation of your blot, particularly if the antigen is very abundant. If cross-reactivity is a problem, load less protein or reduce the amount of antibody.

• One secondary antibody must be labeled with IRDye 680RD secondary antibody, IRDye 680LT secondary antibody, or VRDye secondary antibodies. The other must be labeled with IRDye 800CW secondary antibody or VRDye secondary antibodies.

• It is generally recommended that the IRDye 800CW secondary antibody (800 nm channel) be used to detect the lower-abundance protein target and IRDye 680RD secondary antibody (700 nm) to detect the more abundant protein. If using an instrument that can also detect visible fluorescence, use those channels to detect the internal loading control using Revert 520 Total Protein Stain (preferred) or VRDye 490 Secondary Antibodies in the 490 nm channel or VRDye 549 Secondary Antibodies in the 520 nm channel.

• Always use highly cross-adsorbed secondary antibodies for multiplexed detection. Failure to use cross-adsorbed antibodies may result in increased cross-reactivity.

• For best results, avoid using primary antibodies from mouse and rat together in a multiplexed experiment. The two species are so closely related it is not possible to completely adsorb away all cross-reactivity. If there is no other option but to use mouse and rat together, it is crucial to run single-color blots first with each individual antibody to be certain of expected band sizes.

• If possible, the secondary antibodies should be derived from the same host species (e.g., donkey anti-mouse and donkey anti-goat) to eliminate the chance of the secondary antibodies reacting against one another.

• High abundant targets, such as housekeeping proteins, may perform better in the 490 or 520 channels. Consequently, their working range can be identified and combined with lower abundant targets in the 700 and 800 channels using VRDye secondary antibodies.

General Tips

• Store the IRDye secondary antibody vials at 4 °C in the dark. Do not freeze antibodies, as this will affect performance. Minimize exposure to light and take care not to introduce contamination into the vial. Dilute immediately prior to use. If particulates are seen in the antibody solution, mix gently by inversion and centrifuge before use.

• Protect membrane from light during IRDye secondary antibody incubations and washes.

• Use gels that contain the narrowest well size possible to minimize load volume and concentrate the target protein.

• The best transfer conditions, membrane, and blocking agent for each experiment will vary, depending on the antigen, sample type, and antibody.

• For proteins <100 kDa, the recommended transfer buffer is Tris-glycine buffer with 20% methanol and no SDS. Addition of SDS to the transfer buffer can greatly reduce binding of transferred proteins to the membrane (for both PVDF and nitrocellulose). For proteins >100 kDa, decrease the methanol concentration in the transfer buffer to 10%. For transfers to PVDF membrane, methanol can be eliminated from the transfer buffer.

• Soak the gel in transfer buffer for 5-10 minutes before setting up the transfer. Soaking equilibrates the gel and removes SDS so that it will not be carried over into the transfer tank.

• To maximize retention of transferred proteins on the membrane, allow the membrane to air-dry completely after transfer (approximately 1-2 hours).

• Do not over-block. Extended blocking times, particularly when using nonfat dry milk at 2% or higher, can cause loss of target protein from the membrane (J. Immunol. Meth.122: 129-135, 1989).

Troubleshooting

High background, uniformly distributed

Uneven, blotchy, or speckled background

Non-specific or unexpected bands

Weak or No Signal