Tips for IP-WB with TrueBlot®

Rockland’s TrueBlot® product line is designed to solve the common experimental problems encountered when performing Western blots of Immunoprecipitated (IP) or Co-Immunoprecipitated (Co-IP) samples. The unique detection properties of TrueBlot® secondary antibodies recognize the native (non-reduced) form of IgG over the reduced form of IgG to eliminate interference from the denatured heavy and light chains of the IP antibody. When paired with TrueBlot® IP beads, the TrueBlot® product line allows you to generate reproducible, publication-quality IP/Co-IP-Western blots. Check out the tips below to get the best results when using TrueBlot® products in IP or Western blot assays.

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Tips for Immunoprecipitation Using TrueBlot® Products

• Lysate Sample Preparation

  Prepare cell or tissue lysates using standard methods and be sure all lysate collection steps are performed on ice or at 4°C. Lysis buffers must contain fresh protease and phosphatase inhibitors (such as in cocktail form), as degradation and dephosphorylation can begin immediately upon lysis. For convenience, many manufacturers offer easy-to-use pre-prepared cocktails.

  If preparing your own cocktails, follow the manufacturer’s instructions and recommendations for stock solutions and working concentrations of your specific protease targets, as the half-life of different inhibitors can vary. Additional physical methods can be used in combination with reagent-based lysis methods when desired or necessary.

• Choosing Lysis Buffers

  Various factors influence the choice of lysis buffer, including the location of the protein of interest (i.e., cytosolic, membrane-bound, or within subcellular structures such as the nucleus or mitochondria), solubility of the protein in different detergent types, and compatibility of the buffer in the downstream IP application. Ideally, lysis buffer should be robust enough to extract a sufficient amount of protein, yet provide conditions that retain the native structure of the protein of interest and not disrupt specific protein-protein interactions.

  Radioimmunoprecipitation (RIPA) buffer and NP-40 buffer are common lysis buffers and mostly differ based on the type of detergents used for extraction and solubilization of proteins. In addition to altering the concentration and type of detergents in lysis buffer, other buffer components, such as the concentration of salt, divalent cations, and EDTA, as well as pH, can be altered to achieve the most efficient extraction of the target protein.

  • RIPA Lysis Buffer: Traditional RIPA buffer contains the ionic detergents SDS and sodium deoxycholate, in addition to the non-ionic detergent NP-40. RIPA buffer is very effective for the extraction of various types of protein targets and typically yields an overall lower background. However, RIPA has been demonstrated to denature kinases (Poirier, F. et al., 1982) and the concentration of harsh detergents in the buffer, such as SDS, should be taken into consideration when performing IP, as this may disrupt native conformation of certain proteins or desired protein-protein interactions in Co-IP.
    
    
  • NP-40 Lysis Buffer: NP-40 lysis buffer contains the nonionic detergent NP-40 and is absent of any ionic detergents making it gentler than RIPA, and therefore, less likely to denature kinases and disrupt native protein conformation and protein-protein interactions. However, due to the absence of ionic detergents, NP-40 may not be ideal for efficient lysis and solubilization of nuclear protein targets or other difficult targets and may result in an increased background. NP-40 detergent may be substituted with Triton X-100 when necessary.

• Preclearing Lysates

  A pre-clearing step should be performed by incubating lysate with IP agarose beads in the absence of IP antibody. Preclearing aids in decreasing any non-specific binding to the IP beads that may occur from the various biomolecules contained in the lysate, such as other proteins, carbohydrates, or nucleic acids. An alternative type of pre-clearing step utilizes the addition of a non-specific antibody that is the same species and isotype as the intended IP antibody. In either instance, eliminating non-specific binding to the beads, bead components, or IP antibody should provide an overall decrease in the background and cleaner IP.

• Selecting and Using Primary Antibodies

  Choosing the correct antibody for IP is imperative. Antibodies that have been validated and successfully used in IP should be the first choice, as they can recognize native conformations. If an IP-validated antibody is not available, antibodies that have been successfully used in immunohistochemistry (IHC) are a good alternative to try, as antibodies used in both these methods typically must recognize native epitopes.

  The clonality and amount of IP antibody used also play a role in successful IP procedures. Polyclonal antibodies bind multiple epitopes of a target protein, whereas monoclonal antibodies bind a single epitope; therefore, polyclonal antibodies provide an enhanced retention rate. However, monoclonal antibodies can be used when necessary and can provide better overall specificity and less lot-to-lot variation.

  The amount of IP antibody needed to immunoprecipitate the protein of interest can vary. It is recommended to perform titration in a preliminary experiment to determine the optimal amount. In general, 1–10 µg of IP antibody/10⁷ cells/1 mL lysate is a good starting point. Typically, 2 µg is a sufficient amount of antibody to maximally immunoprecipitate most antigens in 1 mL of extract from 1 x 10⁷ cells. When using TrueBlot® secondary antibodies, it is not recommended to use more than 10 µg (per mL) or a final of 5 µg of IP antibody per lane. Using as little IP antibody as possible minimizes potential contamination of SDS-reduced sample with non-reduced immunoprecipitating antibody chains.

• Selecting Immobilized Beaded Supports

  Selecting the correct IP beads is an essential step in the IP procedure. When using IP antibodies from mouse, rabbit, or goat host species, we recommend using our TrueBlot® Ig IP beads, which are available in either agarose or magnetic formats to achieve the best results. If using Protein A or Protein G IP beads, consult a binding table that lists the expected binding reactivities of Protein A and Protein G to various species and subtypes to ensure the IP antibody is compatible with your choice of bead. For example, mouse IgG1 does not bind well to Protein A but binds strongly to Protein G. Please note, we do not recommend the use of Protein A or G beads when using a rabbit host IP antibody and Rabbit TrueBlot secondary antibodies, as their use can result in non-specific bands.

  Some general considerations for working with TrueBlot IP beads are listed below:

  • When using magnetic beads, gently vortex to resuspend the beads. Do not centrifuge during collection steps. Instead, use a magnetic separator. When using agarose beads, we recommend the vial be inverted several times to get the beads into suspension and ensure homogeneity, as the beads are in suspension and will settle upon storage. Do not vortex agarose beads vigorously, particularly prior to the elution step.
  • Do not freeze the agarose or magnetic beads or allow the beads to dry out. For storage of the opened vials, we recommend that the vial caps be sealed with parafilm to help prevent evaporation of the storage buffer. Storage for both bead formats should be at 4°C.
  • Use a wide-bore pipet tip or cut the end of a pipet tip when pipetting the agarose bead slurry to avoid damage to the beads and assure delivery of the proper bead volume. Magnetic beads can be pipetted using regular tips.

• Binding, Wash, and Elution Buffers

  Note: In the instance that cell lysis buffers are used in the wash or binding steps, the components and concentrations of components in the lysis buffer should be taken into consideration. When harsher detergents such as SDS or deoxycholate are present, the native structure of some proteins or any desired protein-protein interactions may be disrupted in Co-IP.

  • Binding buffers: The binding steps of IP rely on the interactions to form the immune complex (antibody-antigen complex), as well as the interactions between the immune complex (via the IP antibody) to the bead-conjugate (i.e., Protein A, Protein G, or a specific Ig). Thus, IP binding buffers that promote conditions where both affinity interactions are obtained should be used. Forming and maintaining of these interactions generally occur in buffers of relative physiological pH and ionic strength; therefore, 1X PBS or 1X TBS can be used as a baseline to identify the most efficient binding buffer.
    
    
  • Wash buffers: Wash buffers must preserve the specific affinity interactions needed for the IP but efficiently remove any non-specific affinity interactions. The addition of gentle detergents such as NP-40, Triton™ X-100, or CHAPS could be added up to 1% to 1X PBS or 1X TBS to remove background caused by non-specific binding. Altering the NaCl concentration to increase the ionic strength of the buffer may also help to remove stubborn background; however, it is imperative that the specific affinity interactions of the IP remain strong under increased salt conditions.
    
    
  • Elution buffers: For analysis of immunoprecipitated proteins by Western blot using TrueBlot® secondary antibodies, elution of the proteins directly in reducing SDS-PAGE sample loading buffer is convenient and ideal. However, in some instances, the harsh conditions of this buffer combined with a heating step can cause stripping of the subunits of Protein A or Protein G from the IP bead supports and result in non-specific bands in Western blots. In this instance, using the corresponding TrueBlot® Ig IP beads may help remove this contamination. If a gentler elution buffer is needed, 0.1 M glycine, pH 2.0–3.0 may serve as an effective alternative. If using this low pH method, be sure to immediately add Neutralization Buffer (1M Tris-HCl, pH 8.0) prior to loading your gel. Please note that some proteins may not be compatible with low pH methods.

• Incubation

  Incubations should be done at 4°C on a rotator to allow continuous, gentle mixing. The appropriate inhibitors should also be present in the incubation reaction. Typical incubation times can range from 1 hour to overnight; however, optimal incubation times should be determined by the end-user. Choose the method and incubation time that allows for the most effective yield of the protein of interest, while keeping non-specific binding low.

• Recommended Controls for IP experiments

  In addition to standard input (cell lysate only, no IP antibody or IP beads), we recommend running the following controls in tandem with your IP reaction and standard input, especially when first establishing experimental conditions:

  • IP Beads + Cell Lysate (no IP antibody)
    
    
  • Mock IP (lysate with pre-immune serum or normal IgG control from the same host/isotype as the primary antibody, plus beads)

Bonus IP Tips:

1. When performing wash steps, be sure to remove the supernatant completely without disturbing the IP beads.

2. Save the supernatant from the completed binding steps, as well as the washes performed in the IP steps. In the case of a negative result, these samples can be visualized on a western blot to identify any loss of the protein of interest at a specific step in the procedure.

Tips for Western Blotting Using TrueBlot® Products

• Preparation of IP Samples for Western Blot

  As discussed in the elution buffer section, IP samples can be prepared for Western blot by adding reducing SDS-PAGE sample loading buffer directly to the IP beads, followed by heating at 90–100°C for 10 minutes. When using TrueBlot®, it is essential that the immunoprecipitate is completely reduced. The recommended final concentrations for reducing agents are 50 mM DTT or 2% β-Mercaptoethanol (v/v) and can be modified as necessary when optimized by the end-user.

• Blocking Buffer

  The recommended blocking buffer for HRP-conjugated TrueBlot® secondary antibodies is 5% Blotto in 1X TBS-T. When using Fluorescent TrueBlot® secondary antibodies, use Rockland’s Blocking Buffer for Fluorescent Western Blotting (MB-070) or 5% Blotto in 1X TBS (no Tween®). The optimal blocking buffer for Fluorescent TrueBlot® should be optimized by the end-user.

• TrueBlot® Secondary Antibody Selection

  Choosing the appropriate species: TrueBlot® secondary antibodies recognize the IgG of the primary host species. For example, if the host species of your primary antibody is mouse, use Mouse TrueBlot® secondary antibodies.

  Choosing the reporter molecule: TrueBlot® secondary antibodies are available conjugated to a variety of reporter molecules, including horseradish peroxidase (HRP) and fluorescent and near-IR dyes. The choice of reporter molecule in an experiment depends on many factors, such as the need for qualitative or quantitative data, abundance of protein targets, the need to multiplex, and availability of instrumentation and resources.

  Download: Selecting TrueBlot® Products Flowchart (PDF)

• Recommended Dilution

  The recommended dilution for TrueBlot® secondary antibodies is 1:1000 in blocking buffer. The optimal dilution factor should be determined by the end-user. Please note, 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) for your desired application.

• Recommended Controls for Western Blot of IP-Western Procedure

  • Positive control: Species-dependent IgG TrueBlot® will detect SDS-denatured, non-reduced species-specific IgG. A 20–50 ng sample of non-reduced, immunoprecipitating antibody can be included in the immunoblot as a positive control to ensure positive performance of TrueBlot®.
  • Negative control: Samples containing 0.5–2.0 µg 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 immunoprecipitating antibodies.
  • Secondary antibody only: (absence of primary antibody) will detect any non-specific binding of the secondary antibody to cell or tissue lysates.

Bonus Tips:

General Considerations for Fluorescent Western Blotting with Fluorescent TrueBlot® Secondary Antibodies

1. Filter buffers to prevent speckles, as this will remove any precipitates or particles that may be detected.
2. If marking or labeling the membrane, use a pencil as most inks will fluoresce.
3. The bromophenol blue dye present in the SDS-PAGE sample loading buffer can fluoresce and contribute to background. To eliminate fluorescence interference from the dye-front, run the bromophenol blue off the gel or cut from the blot prior to transfer.
4. Be sure to protect the blot from light during incubation and wash steps by using a Western blot box or tray designed to block out light or by covering the blot container with aluminum foil.

References

• Poirier, F., Lawrence, D., Vigier, P., & Jullien, P. (1982). A ts T mutant of Schmidt Ruppin strain of Rous sarcoma virus restricted at 39.5 C for the morphological transformation and the tumorigenicity of chicken embryo fibroblasts. International journal of cancer, 29(1), 69-76.

• Greenfield, E. Antibodies: A Laboratory Manual. 2nd ed., Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 2014.