Far-Western Blotting

Specialized far-Western Analysis

By creative design of bait protein variants and other controls, the far-Western blotting method can be adapted to yield very specific information about protein:protein interactions. For example, Burgess, et al. used a modified far-Western blotting approach to determine sites of contact among subunits of a multi-subunit complex. By an “ordered fragment ladder” far-Western analysis, they were able to identify the interaction domains of E. coli RNA polymerase ß subunit. The protein was expressed as a polyhistidine-tagged fusion, then partially cleaved and purified using a Ni2+-chelate affinity column. The polyhistidine-tagged fragments were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The fragment-localized interaction domain was identified using a 32P-labeled protein probe.

Importance of Native Prey Protein Structure in far-Western Analysis

Far-Western blotting procedures must be performed with care and attention to preserving as much as possible the native conformation and interaction conditions for the proteins under study. Denatured proteins may not be able to interact, resulting in a failure to identify an interaction. Alternatively, proteins presented in non-native conformations may interact in novel, artificial ways, resulting in “false positive” interactions. The prey protein in particular is subjected to preparative processing steps for far-Western blotting that can have significant effects on detection of protein:protein interactions. This is not to imply that identification of valid interactions is not possible but only to stress the importance of appropriate validation and use of controls.

Critical Steps in Far-Western Analysis

Gel Electrophoresis

Separation of proteins by SDS-PAGE (i.e., denaturing conditions with or without a reducing agent) offers more information about MW, presence of disulfides and subunit composition of a prey protein, but may render the prey protein unrecognizable by the bait protein. In these cases, the proteins may need to be subjected to electrophoresis under native conditions, i.e., nondenaturing and without reducing agent.

Transfer to Membrane

After separation on the gel, proteins are electrophoretically transferred from the gel to a membrane in 2-16 hours. The type of membrane (e.g., nitrocellulose or PVDF) used for the transfer of proteins is critical, as some proteins bind selectively or preferably to a particular membrane. The efficiency and rate of protein transfer is inversely proportional to the molecular weight of the protein. In some cases, transfer conditions alter the shape of the protein and destroy or sterically hinder the interaction site on the protein. For far-Western analysis, it is essential that at least the interaction domain of the prey protein is not disrupted by the transfer or is able to re-fold on the membrane to form a three-dimensional (3-D) structure comprising an intact interaction site. Generally, a significant percentage of the protein population renatures upon removal of SDS. When SDS is eliminated during the transfer process, transferred proteins generally renature with greater efficiency and are therefore more easi
ly detected by far-Western blotting. In the event that the protein is unable to re-fold to create an intact binding site, it may be necessary to add a denaturation/renaturation step to the procedure or to perform the protein:protein interaction in-gel without transfer (See In-Gel far-Western Detection below). Denaturation/renaturation is typically accomplished using guanidinium hydrochloride.

Blocking Buffer

After transferring proteins to the membrane, Western blotting procedures require that unreacted binding sites on the membrane be blocked with a non-relevant protein solution. In addition to blocking all remaining binding sites on the membrane, a blocking buffer reduces nonspecific binding and aids in protein renaturation during the probing procedure. A variety of different protein blockers may be used, and no one blocking protein solution will work for all blotting experiments. Any given protein blocker may cross-react or otherwise disrupt the specific probing interaction being studied. Determination of an effective blocking buffer must be made empirically. Often, bovine serum albumin (BSA) is used as a starting point for many membrane-probing reactions. Insufficient blocking may result in high background, whereas prolonged blocking could result in a weak or masked signal. Renaturation of the protein also appears to occur during the blocking step so it is important to optimize the blocking conditions to obtai
n the best signal-to-noise ratio for each application and then not deviate from the method.

Binding and Wash Conditions

Protein:protein interactions vary depending on the nature of the interacting proteins. The strength of the interactions may depend on the pH, salt concentrations and the presence of certain co-factors during incubation with the bait protein. Some protein:protein interactions may also require the presence of additional proteins. Whatever the necessary conditions, they will need to be maintained throughout the procedure to maintain the interaction until it can be detected. This may influence the formulation of washing buffer used between probing steps.

Detection Methods

Depending on the presence of a label or tag on the bait protein, one of four detection methods is used to detect far-Western blot protein:protein interactions:

Direct detection of prey protein with a radioactive bait protein

Indirect detection with antibody to the bait protein

Indirect detection with antibody to the tag of a fusion-tagged bait protein

Detection with biotinylated bait protein and enzyme (HRP/AP) labeled with avidin or streptavidin

Each method has its own advantages and disadvantages.

Several methods are used to generate radioactive isotope labels on bait proteins. The isotope 32P is commonly used to label fusion-tagged protein probes at phosphorylation sites on the tag. This method of phosphorylation has little effect on the protein:protein interaction because the phosphorylation site is located in the fusion tag portion of the protein. Another radioactive method involves direct labeling of bait protein using endogenous phosphorylation sites. However, this technique can only be used if 32P labeling of these sites does not interfere with protein:protein interactions.6 Radioactive detection has also been used when probes are made by incorporation of 35S-methionine during in vitro translation. One disadvantage of this method is that it can only be used for protein probes that have multiple methionine or cysteine residues. Although radioactive isotopes generally do not interfere with interactions because they only alter MW by a few atomic mass units, isotopic detection methods have several di
sadvantages including health hazards and disposal issues.

GST-tagged or polyhistidine-tagged recombinantly expressed bait proteins are often detected with a primary or enzyme-labeled antibody specific to the tag. Antibodies to both these popular fusion tags are commercially available. When recombinant techniques cannot be used to create fusiontagged bait proteins and bait-specific antibodies are not available, bait proteins can be biotinylated and detected with labeled avidin or streptavidin. Pierce offers a full line of biotinylation reagents and enzyme-labeled avidin and streptavidin (refer to the Protein Detection section). Although lysate containing the bait protein can be used for probing membranes, this can result in high background (low signal-to-noise); therefore, it is preferable to purify the bait protein before probing.

Whatever the method of non-isotopic labeling used, the last probing step usually involves use of an antibody or streptavidin probe that is conjugated (labeled) with an enzyme whose localized activity on the membrane can be detected by incubation with a suitable colorimetric, chemiluminescent or fluorogenic substrate. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most popular enzyme labels used for this purpose, with HRP being the most versatile. As with traditional Western blotting, sensitivity in far-Western blotting depends largely on the enzyme substrate system used for detection. Patented SuperSignal Chemiluminescent Substrate Technology enables unmatched sensitivity and lower limits of detection for HRP-based conjugate.

Controls

When identifying protein:protein interactions by the far-Western technique, it is important to always include appropriate controls to distinguish true protein:protein interaction bands from nonspecific artifactual ones. For example, experiments involving detection with recombinant GST fusion proteins should be replicated with GST alone. A bait protein with a mutation in the predicted interaction domain can be processed as a control to determine specificity of the protein:protein interaction. A non-relevant protein can be processed alongside the prey protein sample to act as a negative control. Ideally, the control protein would be of similar size and charge to the protein under investigation and would not interact nonspecifically with the bait protein.

In approaches that use a secondary system for detection of the prey protein, such as enzyme-labeled streptavidin with a biotinylated bait protein, it is important to include a duplicate control membrane that is probed only with the labeled streptavidin. This would reveal any bands resulting from endogenous biotin in the sample or nonspecific binding of the labeled streptavidin. When a fusion tag is used with a corresponding antibody, it is critical to probe one of the control membranes with the labeled antibody alone. This control helps to confirm that the relevant band is not due to nonspecific binding of the labeled secondary antibody. To obtain meaningful results, appropriate test and control experiments should be subjected to gel electrophoresis, transfer and probing in parallel.

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In-Gel far-Western Detectioundefined

Advantages of In-Gel Detection

Because of restrictions associated with the transfer process, blocking, and the possibility of nonspecific binding of bait proteins to unrelated bands on the membranes, it is sometimes advantageous to perform far-Western detection within the gel. In this procedure prey protein samples are separated in precast gels using either native or denaturing conditions. Following electrophoresis, the gels are pre-treated with 50% isopropyl alcohol and water to remove SDS from the gel and allow the prey protein to renature. The gel is then incubated with the bait protein (usually in the pure form). If the bait protein is biotinylated, it is subsequently detected with streptavidin-HRP and a highly sensitive formulation of Pierce’s patented SuperSignal Chemiluminescent Substrate. If the bait protein is fusion-tagged, detection is with an anti-tag HRP-conjugated antibody and the chemiluminescent substrate.

The same controls and experimental conditions necessary for optimization of membrane-based far-Westerns apply to in-gel detection. With in-gel detection the blocking step can be eliminated but the “bait” protein and the labeled detection protein must be diluted in the blocking buffer to reduce nonspecific binding. Also, higher amounts of prey and bait proteins are often required for detection compared to membrane detection with the equivalent chemiluminescent substrate.

ProFound Far-Western Protein:Protein Interaction Kits

Pierce provides two kits for far-Western analysis. These kits are optimized for detection both onmembrane or in-gel. One kit allows the detection of biotinylated bait proteins  and the other allows for the detection of GST-tagged bait proteins . Both kits include blocking and wash buffers, HRP-labeled detection protein (Streptavidin-HRP or Anti-GST- HRP) and an extremely sensitive formulation of UnBlot Chemiluminescent Substrate optimized for both on-membrane and in-gel use. Figure 6 illustrates the general strategy for far-Western analysis. For more detail on these kits, refer to the product section.

Figure 6. General principle of far-Western analysis. The ProFound Far-Western Protein:Protein Interaction Kits follow the non-radiolabeled bait path.