Many protein detection methods, including Western blotting, flow cytometry, ELISA, and immunohistochemistry, require the use of target-specific probes, such as antibodies, that are detected via two-step indirect detection using chemically or fluorescently tagged anti-species antibodies. For example, detection of unlabeled immunoprecipitates via Western blotting is often accomplished using a primary antibody specific to the protein of interest followed by an anti-species secondary antibody conjugated to horseradish peroxidase (HRP).
Interpreting the results from such detection techniques can be difficult because of the presence of additional Western blot signals, caused by cross-reactivity of the secondary antibody with the heavy and light chains of either the endogenous immunoglobulin in total cell lysates or the immunoprecipitating immunoglobulin. Careful experimental design and use of preadsorbed antibodies can reduce these ambiguous results; however, biotin/streptavidin (SA) detection pairs provide an alternative method that addresses these challenges.
Figure 1 – Use of biotin/streptavidin pairs can improve detection of target proteins by eliminating the cross-reactivity of secondary detection antibodies with the heavy and light chain bands present following traditional immunoprecipitation. The cross-reactive interference arises due to binding of the secondary detection antibody (an anti-mouse Ig, in this case) to the mouse-derived immunoprecipitating antibody.
Biotin/streptavidin pairs offer an effective alternative approach to indirect protein detection that avoids the need for secondary anti-species antibodies. In addition, various characteristics of biotin/SA pairs help improve protein purification and detection (Figure 1). First, because biotin is very small (244 Da), its covalent attachment to proteins rarely interferes with function. Its small size also allows for conjugation of multiple biotin molecules on a single antibody, which amplifies signals and increases detection sensitivity. In addition, the binding between biotin and streptavidin is highly specific; it is the strongest known noncovalent interaction (Kd = 10–15 M) between a protein and its ligand. Finally, the complex between these two molecules forms very rapidly and is unaffected by extremes of temperature, pH, organic solvents, and other denaturing agents.
Researchers have the flexibility to customize their own detection panels by using commercially available protein labeling kits, including those for biotinylation. However, because many protocols require buffer exchange prior to and following antibody labeling, the current work flow is time-consuming and subject to significant protein loss at multiple points of sample transfer.
The Amicon® Pro purification system (EMD Millipore, Billerica, MA), an adaptable centrifugal device coupling affinity purification with downstream sample concentration and buffer exchange, offers a solution and makes it convenient to prepare pure, labeled antibody (Figure 2). The system enables highly efficient buffer exchange via diafiltration with simultaneous sample concentration in one 15-min spin. Furthermore, the entire work flow can be performed within a single device, reducing the potential for sample loss.
Figure 2 – The two main components of the Amicon Pro purification system are the large- capacity exchange device and the Amicon Ultra 0.5 mL filter. Notable features of the exchange device include its device cap, which can be labeled and used in the centrifuge instead of the collection tube cap. The tip design of the exchange device connects with the Amicon Ultra 0.5 mL filter for continuous flow/buffer exchange.
This article describes the successful use of the Amicon Pro device for small-scale biotinylation of a target antibody. The biotinylated antibody was used for detection of protein immunoprecipitated from cell lysate.
Materials and methods
Antibody biotinylation: standard protocol
Biotinylation of mouse anti-β-tubulin was performed using Innolink™ Biotin 354S (EMD Millipore). Initial buffer exchange was performed on 50 μg mouse anti-β-tubulin antibody using a centrifugal diafiltration device. The sample was transferred to a new tube, reconstituted Innolink Biotin 354S was added, and the mixture was incubated. The reaction was transferred to a fresh diafiltration apparatus and the buffer was exchanged using three spins with phosphate-buffered saline (PBS) plus sodium azide for each spin. Antibody recovery was determined by measuring absorbance at 280 nm.
Antibody biotinylation using the Amicon Pro purification system
The protocol for biotinylation of mouse anti-β-tubulin was modified for application to the Amicon Pro purification system. The device was assembled with the Amicon Ultra 0.5 mL filter (10K NMWL) attached. Anti-β-tubulin antibody was mixed with PBS containing Innolink Biotin 354S, applied to the Amicon Pro system, and centrifuged. The labeling reaction, now inside the Amicon Ultra 0.5 mL device, was incubated. Next, PBS containing sodium azide was added to the exchange tube of the Amicon Pro system. The unbound biotin was cleared, and biotinylated antibody buffer exchanged by centrifugation. The concentrated, labeled antibody was recovered from the Amicon Ultra 0.5 mL device by reverse spin. Antibody recovery was determined by measuring absorbance at 280 nm.
Immunoprecipitation of native β-tubulin
For each reaction, epidermal growth factor (EGF)-stimulated A431 cell lysate was combined with anti-β-tubulin antibody and incubated overnight with end-over-end mixing. Next, half of this reaction was transferred to the exchange tube of an Amicon Pro device containing prewashed Protein A agarose and incubated with gentle agitation. PBS was added and the filtrate was cleared by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was added to the resin, resuspended by pipetting, and then transferred to a microfuge tube. Following incubation, the beads were returned to the device and centrifuged to collect the filtrate.
Electrophoresis and blotting
Immunoprecipitated samples and control lysate were denatured and loaded onto NuPAGE® Bis-Tris gels (Life Technologies, Carlsbad, CA). Gels were run at 200 V for 40 min. They were then removed from the cassette and equilibrated in transfer buffer supplemented with 10% methanol. After equilibration, the proteins were transferred to Immobilon®-P PVDF membrane (EMD Millipore) using a semidry transfer system (Bio-Rad, Hercules, CA). Blots were briefly rinsed in Milli-Q® water (EMD Millipore) and assembled directly into the SNAP i.d.® 2.0 blot holder (EMD Millipore) for immunodetection.
Once blot holders were placed in the SNAP i.d. 2.0 system, Bløk®-CH blocking buffer (EMD Millipore) was added and the vacuum immediately activated. Primary antibody (mouse anti-β-tubulin or biotinylated anti-β-tubulin, both diluted to 1 μg/mL in blocking buffer) was added to the blot holder and incubated. The vacuum was initiated and blots washed three times with tris-buffered saline with Tween® 20 (TBST) surfactant. After the vacuum was turned off, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody diluted in blocking buffer. The vacuum was activated and blots washed with TBST. Probed blots were visualized with Luminata™ Classico Western HRP Substrate (EMD Millipore). Blots were patted dry, exposed to X-ray film, and developed.
The highly specific binding between biotin and streptavidin makes this molecular pair invaluable for protein detection. In cases in which an antibody must be biotinylated before use, however, the currently available methods for small-scale labeling (≤50 μg) present a major obstacle; the process is tedious and inefficient. It requires initial antibody cleanup, labeling, removal of unbound label/buffer exchange, and concentration of labeled protein. The two buffer exchange steps risk the greatest sample loss and are the most time-consuming for researchers.
Table 1 – Comparison of biotinylation work flows with respect to time and antibody recovery
The overall processing time and recovery efficiency of the Amicon Pro system were compared to a standard protocol provided with the Innolink Biotin 354S reagent (Table 1). The differences would be even more significant if traditional tube or cassette-based dialysis methods were employed, which typically involve overnight incubations, but even so, biotinylation using the Amicon Pro system reduced processing time by 70%.
The improved recovery is most likely a reflection of sample containment within a single device for the entire process. It may also be caused by the gentler method of buffer exchange.To achieve complete buffer exchange, the smaller diafiltration device (0.5 mL) used with the traditional biotinylation protocol required multiple concentration/dilution cycles. These multiple cycles may cause either loss of protein function due to destabilization of the tertiary structure, or physical protein loss due to aggregation and precipitation.
Figure 3 – Both preparations of biotinylated anti-β-tubulin successfully detect β-tubulin in Western blots. Aliquots of EGF-stimulated A431 cell lysate (0.25–4 μg) were resolved by SDS-PAGE, transferred onto Immobilon-P membrane, and probed for β-tubulin in the SNAP i.d. 2.0 system using the various indirect detection pairs. In each case, the same quantity of primary (β-tubulin-specific) antibody was used.
To assess the functional performance of the newly biotinylated antibodies obtained using the two protocols described, their detection ability was compared with that of a species/anti-species β-tubulin detection pair (Figure 3). Both biotin/SA pairs demonstrated the same, if not slightly better, sensitivity than the species/anti-species detection pair.
When species/anti-species antibody pairs are used, interpretation of Western blotting results is often confounded by cross-reactivity of the secondary antibody with immunoglobulin heavy (50 kDa) and light (25 kDa) chains present in either the total lysate or immunoprecipitated fractions. The signals that arise from this cross-reactivity are particularly problematic when the protein(s) of interest are similar in molecular weight to the immunoglobulin chains.
Figure 4 – Biotin/SA detection pairs permit visualization of the β-tubulin signal in Western blots of immunoprecipitated fractions. Total A431 cell lysate and IP fractions were resolved by SDS-PAGE, transferred to membrane, and probed for the presence of β-tubulin. Given that the same antibody was used for both IP and primary detection in immunoblotting, cross-reactivity of the secondary antibody resulted in appearance of bands at 25 and 50 kDa. Of greater concern was that the signal from the heavy chain masked signal expected from β-tubulin, a protein of similar molecular weight.
The ability to detect β-tubulin following immunoprecipitation (IP) was assessed to demonstrate the benefits of employing a biotin/SA pair (Figure 4). In blots probed with the species/anti-species pair, two prominent bands were present in the IP fraction; these correspond to binding of the secondary antibody to the light and heavy chains of the anti-tubulin antibody originally used to capture tubulin from the lysate. When an identical blot was probed with secondary antibody alone, this result was confirmed. In each case, the 50 kDa heavy chain masked the signal expected from the similarly sized β-tubulin protein. In contrast, a single, prominent band was detected in the immunoprecipitated fractions probed with the biotin/SA pair.
This article has demonstrated the advantages of using the Amicon Pro purification system for small-scale antibody labeling. The centrifugal device offered a comparatively streamlined work flow, providing significant time-savings when compared to functionally equivalent platforms. It also led to reduced sample loss, a potentially important consideration given the cost of commercial antibodies.
The study also confirmed the importance of using biotin/SA pairs to eliminate immunoglobulin cross-reactivity. With the flexibility of the Amicon Pro purification system, small amounts of antibody can be labeled quickly and easily with reliable performance and superior yield.
Amedeo Cappione, Ph.D., is Senior Scientist; Masaharu Mabuchi is Research Scientist; David Briggs, Ph.D., is Director Product Marketing; and Timothy Nadler, Ph.D., is Senior R&D Manager, EMD Millipore Corp., 17 Cherry Hill Dr., Danvers, MA 01923, U.S.A.; tel.: 978-762-5007; fax: 978-762-5386; e-mail: firstname.lastname@example.org.