Using IRDye® Subclass-Specific Antibodies for Western Blot Detection

Introduction

This guide will help you understand how to use IRDye® subclass-specific antibodies in your Western blot protocol.

In mice, there are five unique subclasses of IgG:

• IgG₁

• IgG_2a

• IgG_2b

• IgG_2c

• IgG₃

Each subclass is based on small differences in amino acid sequences in the constant region of the heavy (gamma) chains. IRDye subclass-specific antibodies only react with the heavy chain of a primary antibody.

So, antibodies directed against a particular subclass will not recognize antibodies directed against other subclasses. For example, IRDye Goat anti-Mouse IgG₁ recognizes mouse gamma 1; it will not recognize mouse gamma 2a, 2b, 2c, or gamma 3. Because of this, IRDye Goat anti-Mouse IgG₁, IRDye Goat anti-Mouse IgG_2a, and IRDye Goat anti-Mouse IgG_2b allow for two-color detection using primary antibodies derived from mice.

Other IRDye Secondary Antibodies are whole IgG (H + L) and react with the heavy (gamma) and light (kappa or lambda) chains of the primary antibody. Figure 96 demonstrates the differences in detection between IRDye antibodies.

Antibody subclasses may also be designated by their light chains. There are two types of light chains, kappa and lambda. In mice, 95% of light chains are kappa, and 5% are lambda. These subclasses still contain the heavy (gamma) portion of the antibody, so IRDye subclass-specific antibodies still recognize them. If the subclass of the primary antibody is unknown, IRDye whole IgG secondary antibodies may be used since they recognize most mouse IgG subclasses.

Suggested Materials

This section is intended as a guideline; other materials may be substituted, if desired.

• Proteins transferred to a nitrocellulose or PVDF membrane

• Intercept® Blocking Buffer

• 10X PBS

• 20% Tween® 20

• SDS (if using PVDF membrane)

• Suggested mouse primary antibodies for normalization:

  • Beta-Actin Mouse mAb IgG_2b (PN 926-42212)

  • Alpha-Tubulin Mouse mAb IgG₁ (PN 926-42213)

• One or two IRDye® Secondary Antibodies from Table 7

Table 7. IRDye subclass-specific antibodies.

Description	LI‑COR Part Number
IRDye 800CW Goat anti-Mouse IgG1-Specific	926-32350
IRDye 800CW Goat anti-Mouse IgG2a-Specific	926-32351
IRDye 800CW Goat anti-Mouse IgG2b-Specific	926-32352
IRDye 680LT Goat anti-Mouse IgG1-Specific	926-68050
IRDye 680LT Goat anti-Mouse IgG2a Specific	926-68051
IRDye 680LT Goat anti-Mouse IgG2b Specific	926-68052

Multicolor Considerations

Multiple antigens can be detected on the same blot in different ways.

• Using IRDye® subclass-specific antibodies

• Using IRDye whole IgG antibodies visualized in different fluorescence channels (e.g., 700 and 800 nm)

Multiple targets can be normalized on the same blot with a total protein stain, such as Revert™ Total Protein Stain. The included protocol will require modification if you are using Revert. See the product pack insert for details.

Multicolor detection requires careful selection of primary and secondary antibodies. The following guidelines will help with the design of multicolor experiments:

• If the two primary antibodies are monoclonals (mouse) and are IgG₁, IgG_2a, or IgG_2b, IRDye subclass-specific secondary antibodies must be used. The same subclasses cannot be combined in a multicolor Western blot (e.g., two IgG₁ primary antibodies).

• If the two primary antibodies are derived from different host species (for example, primary antibodies from mouse and chicken), IRDye whole IgG secondary antibodies derived from the same host and labeled with different fluorophores must be used (e.g., IRDye 800CW Donkey anti-Mouse and IRDye 680LT Donkey anti-Chicken).

• Before combining primary antibodies in a multicolor experiment, always perform preliminary blots with each primary antibody alone to determine the expected banding pattern and possible non-specific background bands.

Protocol

IRDye® subclass-specific antibodies are easily incorporated into any Western blot protocol. The sample protocol provided below, optimized for LI‑COR reagents, is recommended. After protein transfer to the membrane is complete, perform the following steps for one- or two-color detection.

Step 1. Wet Membrane

For Nitrocellulose Membranes

Wet in 1X TBS or PBS for 5 minutes or until fully hydrated (using the appropriate buffer system).

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 at room temperature with gentle shaking.

Be sure to use sufficient blocking buffer to cover the membrane (a minimum of 0.4 mL/cm² is suggested).

Step 3. Dilute Primary Antibody

1. Use Intercept T20 Antibody Diluent as the primary antibody diluent or prepare your own by adding 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.

   • Depending on the primary antibody, dilutions may range from 1:200 – 1:5,000.
   • Use enough antibody solution to completely cover the membrane.

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.

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.

Step 5. Wash Membrane

1. Carefully pour off primary antibody solution.
2. Rinse the membrane with 1X TBS-T (0.2% Tween 20) or 1X PBS-T (0.2% Tween 20).
3. Cover blot with 1X TBS-T or 1X PBS-T.
4. Shake vigorously on platform shaker at room temperature for 5 minutes.
5. Pour off wash solution.
6. Repeat steps 3 to 5 for a total of 4 washes.

Step 6. 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. The following includes recommendations on antibody dilution buffers.

When performing multicolor detection, dilute your secondary antibodies simultaneously in the same buffer.

Secondary Antibody Diluent for Nitrocellulose Membranes

Use Intercept T20 Antibody Diluent. Alternatively, add Tween® 20 to a final concentration of 0.2% in Intercept® Blocking Buffer. Do not add SDS.

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 7. 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 8. Wash Membrane

Protect membrane from light during washes.

1. Carefully pour off secondary antibody solution.
2. Rinse the membrane with 1X TBS-T or 1X PBS-T.
3. Cover blot with 1X TBS-T or 1X PBS-T.
4. Shake vigorously on platform shaker at room temperature for 5 minutes.
5. Pour off wash solution.
6. Repeat steps 3 to 5 for a total of 4 washes.

Step 9. 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 10. 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.

Example Data

Figure 97 and Figure 98 demonstrate multicolor Western blot detection using IRDye® subclass-specific antibodies (A) and IRDye whole IgG antibodies (B).

• Blot A was probed with mouse anti-PTEN (IgG_2b) and mouse anti-GAPDH (IgG₁) for normalization. The blot was detected with IRDye 800CW Goat anti-Mouse IgG_2b (PN 926-32352) and IRDye 680LT Goat anti-Moust IgG₁ (PN 926-68050).

• Blot B was probed with mouse anti-PTEN (IgG_2b) and chicken anti-GAPDH for normalization. The blot was detected with IRDye 800CW Donkey anti-Mouse (PN 926-32212) and IRDye 680LT Donkey anti-Chicken (PN 926-68028).

• Blot C was probed with mouse anti-PTEN (IgG_2b) and mouse anti-GAPDH (IgG₁) for normalization. The blot was detected with IRDye 800CW Goat anti-Mouse (PN 926-32210) and IRDye 680LT Goat anti-Mouse IgG₁ (PN 926-68050).

Both IRDye whole anti-mouse IgG secondary antibodies and IRDye subclass-specific secondary antibodies recognize the gamma chain of the primary antibody (C), causing detection in both channels. IRDye subclass-specific antibodies can be used in combination with IRDye whole goat anti-rabbit secondary antibodies.

IRDye subclass-specific secondary antibodies should not be used in combination with IRDye whole anti-mouse IgG secondary antibodies for multicolor detection.

Multicolor Western blot detection can be achieved by multiplexing mouse primary antibodies and IRDye subclass-specific antibodies. Figure 99 demonstrates two-color detection utilizing LI‑COR mouse primaries and IRDye subclass-specific secondaries.

• Blot A was probed with Beta-Actin Mouse mAb IgG_2b (PN 926-42212) and p53 mouse mAb IgG_2a. The blot was detected with IRDye 800CW Goat anti-Mouse IgG_2a and IRDye 680LT Goat anti-Mouse IgG_2b.

  p53 expression in HeLa cells is not typically observed due to oncoprotein E6.

• Blot B was probed with Alpha-Tubulin Mouse mAb IgG₁ (PN 926-42213) and ERK2 mouse mAb IgG_2b. The blot was detected with IRDye 680LT Goat anti-Mouse IgG₁ and IRDye 800CW Goat anti-Mouse IgG_2b.