IP-WB with TrueBlot® Protocol

The mouse, rabbit, goat, and sheep-derived TrueBlot® IgG antibodies represent a unique series of respective anti-species IgG secondary antibodies which are conjugated to horseradish peroxidase (HRP). They are designed for use in immunoprecipitation (IP) and Western blot (WB) procedures in which the same species antibody is used for both the IP and immunblotting steps. TrueBlot® secondary antibodies eliminate interference from the denatured/reduced heavy and light chains of the IP antibody by detecting only the native, non-reduced form of IgG. Using TrueBlot® secondary antibodies easily accommodates the detection of immunoprecipitated proteins with overlapping molecular weights to the heavy and light chain to provide accurate, publication-quality IP–Western Blots.

Note: Protocols for TrueBlot® Western blot kits and TrueBlot IP/WB sets can be found on product pages.

Reagents Required

¹RIPA and NP-40 (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1% NP-40) are commonly used lysis buffers. For optimal results, choose a lysis buffer that is most compatible with your protein of interest and downstream IP application. If running protein assays prior to SDS-PAGE, be sure the components of your lysis buffer are compatible with your choice of assay.

²The type of protease and phosphatase inhibitors used, as well as the final working concentration, varies and should be optimized by the end user. However, a suggestion of final concentrations for a general protease inhibitor cocktail is 1.0 mM PMSF, 10 µM leupeptin, 0.1 µM aprotinin, and 1.0 µM pepstatin. The recommended final concentration for phosphatase inhibitors is 1.0 mM Na3VO4 and 1 mM NaF. A variety of cocktails are also available commercially.

³Do not vortex the agarose IP beads prior to the elution step as this may cause damage. Agarose IP beads are in suspension and settle upon storage. Prior to use, mix the vial well by gentle inversion to ensure delivery of the proper bead volume. The Ig IP beads give the best results; however, if using Protein A or G beads with mouse or goat TrueBlot® secondary antibodies, follow manufacturer’s recommended IP protocol. We do not recommend the use of Protein A or G with rabbit TrueBlot®. It is not recommended to use more than 10 μg (per mL) or a final of 5 μg of IP antibody per lane.

⁴Use of standard IP buffers for binding and wash steps, such as 1X PBS or 1X TBS with or without low concentrations of gentle detergent, as well as other IP buffers should be optimized by the end user for their specific immune complex. If using cell lysis buffer in these steps, take into consideration the detergent type and concentration, as these factors may disrupt native structure or protein-protein interactions.

⁵Prepare reducing 2X SDS sample buffer fresh and use within the hour. Discard remainder. Concentration of reducing agents can be increased, if needed.

⁶Choose transfer buffer based on protein of interest, gel type, membrane, and transfer type.

⁷The final TWEEN 20 concentration used in the 1X TBS-T buffer may be adjusted if necessary (0.01–0.1%) when optimized by the end user.

Table 1. TrueBlot® Agarose Ig IP Beads or IP/WB Set

¹IP/WB sets include Ig IP agarose beads and corresponding TrueBlot® secondary antibody (HRP).
Note: TrueBlot® general-use magnetic beads and Ig IP magnetic beads are also available.

Table 2. HRP-conjugated TrueBlot® Secondary Antibodies

²Western blot kits contain the following components: (1) TrueBlot® secondary antibody (50 μL), (2) Enhancer solution (25 mL), (3) Blocker (10 g), (4) 20X Assay buffer (30 mL), (5) HRP Substrate A (12.5 mL), (6) HRP Substrate B (12.5 mL), (7) Primary antibody to be used at end-user's descretion, (8) Western blot incubation tray.

Procedure for Cell Lysate Preparation

1. Aspirate or pipet off cell culture media. Wash with ice-cold 1X PBS, then remove 1X PBS.
2. Harvest approximately 1 x 10⁷ cells by using a cell scraper and transfer to a conical tube. If working with adherent cells, you can skip this step and lyse directly on the plate (see step 5).
   Note:The total number of cells per mL and the cell equivalent loaded per lane of gel should be optimized specifically for each protein and antibody. Alternatively, protein concentration can be determined using a protein assay.
3. Wash cells with ~10 mL of cold 1X PBS and centrifuge at 400 x g for 10 minutes at 4°C.
   Note: If using adherent cells, wash cells on the plate while keeping the plate on ice.
4. Discard the supernatant and repeat step 3.
5. After the second wash, remove the supernatant and resuspend the cell pellet in 1 mL of cold Lysis buffer containing protease inhibitors. The final concentration of cells should be about 1 x 10⁷ cells/mL.
   Note: If using adherent cells, the cold lysis buffer can be added directly to the plate and put on a rocker at 4°C. Harvest by either scraping or collecting just the supernatant. If collecting just the supernatant, proceed to step 8.
6. Gently vortex and transfer to a 1.5 mL microcentrifuge tube.
7. Place on ice for 30 minutes with occasional mixing.
8. Centrifuge at 10,000 x g for 15 minutes at 4°C.
9. Carefully collect the supernatant without disturbing the pellet and transfer to a new, clean 1.5 mL microcentrifuge tube and discard the pellet.
10. The protein concentration can be determined by a protein assay. Samples can be diluted to 1 mg/mL if desired.
11. The cell lysate can be frozen at this point for long-term storage at -80°C.

Procedure for Cell Lysate Preclearing

1. Resuspend the immobilized anti-species-specific IgG bead slurry. Remove 50 μL and wash in lysis buffer or IP buffer, if different. Resuspend in 50 μL of cell lysis buffer or IP buffer. Bead slurry by repeatedly inverting the vial several times. Do not vortex.
2. Add 500 μL of cell lysate (~5 x 106 cells or ~500 μg protein) to the pre-equilibrated bead slurry and incubate on a rocking platform or orbital shaker for 30–60 minutes at 4°C.
3. Centrifuge at 2,500 x g for 2–3 minutes at 4°C and transfer the supernatant to a new 1.5 mL tube. If any of the bead slurry has been transferred, centrifuge again and carefully transfer the supernatant to another fresh 1.5 mL tube.

Procedure for Immunoprecipitation

1. Add 1–10 μg of immunoprecipitation antibody to the tube containing the cold, precleared cell lysate. 
   Note: This concentration of antibody is suggested as a starting point. Each investigator may desire to titrate the concentration of antibody and volume of cell lysate in preliminary experiments to determine the optimal conditions, e.g., 1–10 μg/1 x 10⁷ cells/1 mL lysate.
2. Typically, 2 μg is a sufficient amount of antibody to maximally immunoprecipitate most antigens in 1 mL of extract from 1 x 10⁷ cells. Using as little IP antibody as possible minimizes potential contamination of SDS-reduced sample with non-reduced immunoprecipitating antibody light chain. It is not recommended to use more than 10 μg (per mL) or a final of 5 μg per lane. 
3. Incubate at 4°C for 1 hour on a rocking platform or a rotator. 
4. Add at least 50 μL of pre-equilibrated bead slurry to capture the immune complexes. 
5. Incubate for 1 hour or overnight at 4°C on a rocking platform or a rotator. 
   Note: Steps 1 and 3 can be combined for a single incubation. Centrifuge the tube at 2,500 x g for 30 seconds at 4°C. 
6. Carefully remove the entire supernatant and wash the beads 3–5 times with 500 μL of cold lysis buffer or IP buffer, centrifuging to pellet beads between each wash. To minimize background, be sure to completely remove the supernatant after each wash. 
7. After the last wash, carefully aspirate the supernatant and add 50 μL of SDS reducing sample loading buffer or equivalent to the bead pellet. 
   Note: 1X or 2X SDS reducing sample buffer can be added to the beads. In-house experiments use 2X. Please take into consideration the composition of the loading buffer. It is important that the sample is completely reduced.
8. Gently vortex and heat samples at 90–100°C for 10 minutes. 
9. Centrifuge at 10,000 x g for 5 minutes, carefully collect the supernatant, and load onto the gel. 
10. Alternatively, the supernatant samples can be collected, transferred to a clean 1.5 mL microcentrifuge tube and frozen at -80°C, if the gel is to be run at a later time. 
11. Follow manufacturer’s instructions for SDS-PAGE. 

Procedure for Western Blot

1. Following SDS-PAGE, transfer proteins from the gel onto either a PVDF or nitrocellulose membrane. 
   Note: For best protein transfer results, follow the instructions provided by the transfer system manufacturer.
   Optional: To determine whether the proteins have been transferred to the membrane, stain with a 0.1% Ponceau S solution. Protein bands can be visualized after staining for 5 minutes. To remove the Ponceau S stain, rinse with distilled water or 1X TBS-T until most of the dye is removed before moving on to blocking step. Residual dye will not affect subsequent steps.
2. Block the membrane with 5% BLOTTO solution (w/v) and incubate for 2 hours at room temperature or overnight at 4°C on a rocking platform. 
   Note: It is recommended to use Milk or BLOTTO as the blocking reagent for HRP-conjugated TrueBlot® secondary antibodies, as BSA may not block the reduced chain as effectively.
3. Prepare the primary immunoblotting antibody in blocking buffer as recommended by the supplier. 
   Note: If the recommended concentration is not known, use a standard concentration of 1–2 μg/mL. If using hybridoma tissue culture supernatant or serum for immunoblotting, preliminary experiments should be performed to evaluate whether dilution of the supernatant or serum is needed for best results.
4. Incubate the blot with primary antibody for at least 2 hours at room temperature or overnight at 4°C on a rocking platform. 
   Note: Specific times should be determined empirically for optimal results.
5. After the incubation of the membrane with the primary antibody, wash the blot at least 3–5 times in 1X TBS-T, each wash for a minimum of 5–10 minutes each. Total should be more than 1 hour. 
6. Prepare the TrueBlot® secondary antibody at a 1:1000 dilution in the blocking buffer. 
   Note: This dilution is intended as an initial recommendation. Specific conditions for each protein and antibody combination should be specifically optimized by the end user. To optimize the detection of mouse IgG1, we recommend performing a dot blot or titration to determine the ideal dilution factor (starting at 1:1000).
7. Incubate the blot with TrueBlot® secondary antibody for 1 hour at room temperature on a rocking platform.
   Note: Specific times should be determined empirically for optimal results.
8. Wash the blot at least 3–5 times in 1X TBS-T, each wash for at least 5 minutes each. Total should be more than 1 hour. 
9. Develop the blot using a chemiluminescent HRP substrate following the manufacturer’s instructions. 
10. Image the blot with an imaging system compatible with chemiluminescent detection or by exposing the blot to x-ray film.

Notes:

Recommended positive control: Species-dependent IgG TrueBlot® will detect SDS-denatured, non-reduced species-specific IgG. A 20–30 ng sample of non-reduced, immunoprecipitating antibody can be included in the immunoblot as a positive control to ensure positive performance of TrueBlot®.

Recommended negative control: Samples containing 0.5–2.0 ug of reduced species-specific IgG (prepared and run immediately as described in Sample Preparation) can be included as a negative control to ensure that individual TrueBlots® do not detect heavy and light chains of the immunoprecipitating antibodies.

Recommended additional controls:

1. Omit the cell extract during the IP
2. Omit the IP antibody during the IP
3. Omit the immunoblotting antibody

TrueBlot® Troubleshooting Guide

Table 3. Common Troubleshooting Techniques for TrueBlot® Products