PRODUCT APPLICATION FOCUS

Antibody-free method for detection on blots using enzyme fragment complementation

Joe Horecka, Neil W. Charter, Betty L. Bosano, Peter Fung, Phil Kobel, Kun Peng, and Richard M. Eglen

BioTechniques 40:381-383 (March 2006) doi 10.2144/000112119

Characterization of protein expression and high complemented were incubated for 1 h with EA reagent expression in recombinant systems enzyme activity have been created (DiscoveRx) to enable complemen- has been enhanced through the avail- and are known as ProLabel™ (PL; tation with the membrane-bound PL. ability of techniques such as Western SSNSLAVVLQRRDWENPGVTQLN Chemiluminescent β-gal substrate blot analysis and enzyme-linked RLAAHPPFASWRNSEEARTDRPS (DiscoveRx) was then added directly to immunosorbent (ELISA) (1). QQLRSLNGE; DiscoveRx, Fremont, the EA solution in which the blot was A limitation of these techniques is the CA, USA) (5). Since EA essentially immersed. Since noncomplemented EA need for an antibody specific to the lacks β-gal activity in the absence of is inactive, it is not necessary to remove protein of interest. The development of PL, EFC technology is ideally suited or wash excess EA from the membrane. epitope tags such as c-myc and HA has for use in homogenous assays that can After a 15-min incubation, the blots addressed this limitation by providing specifically and quantitatively measure were drained of excess liquid, placed the means to characterize recom- PL availability for complementation between sheets of clear plastic, and binant without the need for a (5–7). The novel blot assay extends this the β-gal chemiluminescent activity protein-specific antibody (2,3). While technology to the detection of recom- was detected using a digital imaging they provide good utility, techniques binant proteins on a solid support, such system. such as Western blotting involve as a nitrocellulose or polyvinylidine A 5-min exposure produced suffi- multiple incubation and wash steps that difloride (PDVF) membrane. cient signal to detect NFAT-PL in a cell require a significant amount of time to To demonstrate the utility of the EFC lysate sample equivalent to 4 μg total perform. Thus, there exists a need for blot assay, we examined its ability to protein (Figure 1). A major band with a more efficient method to detect and detect recombinant proteins in extracts an apparent molecular weight of 85 characterize recombinant proteins. prepared from CHO cells transfected kDa was detected, which is consistent We have developed an alternative blot with a variety of PL-tagged constructs. with the expected mass of the NFAT- assay, which provides similar utility These examples include a stably trans- PL fusion protein (86.5 kDa). Two to Western blotting without the need fected cell line expressing the nuclear minor bands were also detected: one for specific antibodies or multiple factor of activated T cells protein of approximately 60 kDa is consistent incubation and wash steps (Table 1). tagged at its C terminus with ProLabel with it being a proteolytic cleavage The ability to detect recombinant (NFAT-PL) (8,9) as well as transiently product of full-length NFAT-PL, and proteins using this novel blotting transfected cells expressing c-Jun- the other of approximately 6 kDa is technique takes advantage of enzyme PL and cyclin D-PL fusion proteins. consistent with it being the PL peptide fragment complementation (EFC), Proteins extracted from cell lysates moiety (55 amino acids, approximately whereby a short peptide (the α were separated by sodium dodecyl 6.3 kDa) liberated by proteolytic fragment or enzyme donor) derived sulfate polyacrylamide gel electropho- cleavage. Transiently expressed c-Jun- from the N terminus of β-galacto- resis (SDS-PAGE) and transferred to a PL and cyclin D-PL of approximately sidase (β-gal) can reconstitute enzyme nitrocellulose membrane using standard 45 and 42 kDa were detected in cell activity in a corresponding deletion techniques. An additional fusion lysates equivalent to 0.5 and 0.35 μg mutant of full-length β-gal (the enzyme protein consisting of NeutrAvidin™ total protein, respectively. Control cell acceptor or EA) (4). Variants of the α chemically conjugated to PL (NA-PL) extracts from CHO cells transiently fragment optimized for recombinant was included as a control. The blots transfected with vector only lacked

DiscoveRx Corporation, Fremont, CA, USA

Vol. 40, No. 3 (2006) BioTechniques 381 PRODUCT APPLICATION FOCUS all bands. The NA-PL control sample in loading was likely to be minimal. equivalent in cost to conventional exhibited three major bands: the 22 and Using identical exposure times to reagents. While the 27 kDa bands are consistent with the capture chemiluminescent signal, both assay has been optimized for use expected molecular weights of a neutra- the EFC and Western blot techniques with the substrate provided in the kit vidin monomer conjugated with one or were able to detect NFAT-PL in lysate (commercialized as the EAstern™ two PL peptides, respectively, and the containing 0.25 μg total protein (Figure blot assay; DiscoveRx), it is possible 5-kDa band is consistent with the free 2). For the anti-NFAT antibody, pilot to use other β-gal substrates, although unconjugated PL peptide used in the titration experiments were required to assay sensitivity and exposure times synthesis (47 amino acids, approxi- determine the concentration required may vary considerably. In experiments mately 5.5 kDa). There is likely to be to provide sensitivity equivalent to that where loading controls are required, a little limitation in the size of potential obtained by EFC blot. While sensitivity number of options are available that are fusions detected by EFC blot, since low was comparable, a higher background compatible with EFC blots. Reversible molecular weight PL is readily detected was associated with the Western blot in both the NA-PL control and in the compared with the EFC blot. This was inferred cleavage products of other primarily due to nonspecific interac- full-length recombinant proteins. tions of the primary antibody at the high The detection sensitivities of EFC concentrations used. Similar results and Western blotting were compared were seen in side-by-side EFC/Western using a protein blot containing a blot comparisons with a number of duplicate titration of cell lysate and, other PL-tagged fusion proteins and a consequently, NFAT-PL protein. The variety of monoclonal and polyclonal blot was divided into two and probed antibodies (data not shown). This either by EFC as described above or by experiment also demonstrates that Western blot analysis, using an anti- EFC blotting is entirely exchangeable NFAT monoclonal antibody followed with the current protocols for Western by a horseradish peroxidase (HRP)- blotting, since both methods use the conjugated secondary antibody and same and blotting HRP chemiluminescent substrate. Since steps. the samples and transfer conditions EFC blot reagents have a shelf were identical for both blots, variation life of at least 6 months and are

Table 1. Comparison of EFC and Western Blot Protocols Figure 1. Enzyme fragment complementa- Time EFC Blot Western Blot tion (EFC) blot detection of ProLabel (PL)- (h) tagged fusion proteins in cell lysates. CHO-K1 cells stably transfected with DNA encoding the nuclear factor of activated T cells protein tagged 1 at its C terminus with ProLabel (NFAT-PL) were SDS-PAGE of PL-tagged protein sample and transfer to blot lysed at 4 × 106 cells/mL in cell lysis buffer 2 [phosphate-buffered saline (PBS), 0.5% CHAPS, 1× Complete Mini EDTA-Free Protease Inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, Brief rinse with water USA)]. Lysates of CHO-KI cells transiently 3 Add 5 mL chemiluminescent Incubate with blocking buffer transfected for 2 days with DNA encoding c- Jun-PL, cyclin D-PL, and vector only were also substrate and incubate (15 min) generated. Four micrograms NFAT-PL and 0.5 μg c-Jun-PL, 0.35 μg cyclin D-PL, or 0.5 μg Add 5 mL chemiluminescent Incubate with the primary antibody CHO-KI lysates were loaded onto a 4%–20% so- 4 substrate and incubate (15 min) dium dodecyl sulfate (SDS) polyacrylamide gel, separated by electrophoresis in MES buffer, and Acquire image (5–20 min) transferred to a nitrocellulose membrane. NA- PL control protein was run in an adjacent well. 5 Wash six times for 10 min each Detergent and other components were removed from the resulting blot by rinsing in ultrapure wa- ter for 2 min. The blot was incubated with 5 mL 6 Incubate with the secondary antibody enzyme acceptor (EA) reagent for 1 h at room temperature with rocking. Five milliliters chemi- luminescent substrate were added, and the blot 7 Wash six times for 10 min each was incubated for a further 15 min. Excess liquid was drained, and the blot was placed between two Incubate with detection reagent (5 min) sheets of clear plastic. The signal resulting from 8 the complementation of EA with membrane- Acquire image (5–60 min) bound PL was detected using an Epi Chem II Station digital imaging system (UVP, Upland, EFC, enzyme fragment complementation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel CA, USA). Data shown is an inverted image electrophoresis; PL, ProLabel; EA, enzyme acceptor. acquired with a 5-min exposure. 382 BioTechniques Vol. 40, No. 3 (2006) protein stains, such as Ponceau S, can not dependent on the affinity of the binant protein levels at any point during be used to compare total protein levels antibody for the protein in question. protein expression and purification. on a blot prior to EFC analysis. More Thus, in some cases, EFC blotting may rigorous analysis such as determination provide greater sensitivity, particu- of actin or tubulin levels would require larly when high affinity antibodies COMPETING INTERESTS Western blotting. In our experience, a are not available. Second, since EFC STATEMENT blot in which EFC analysis has been blotting is based on enzyme fragment performed can be washed to remove the complementation, there is virtually The authors are employed by chemiluminescent substrate and used none of the problematic background DiscoveRx Corporation, which will directly for Western analysis. Blots often associated with Western blotting market the assay as a commercial can also be stored at 4°C in phosphate- that can be caused by binding of the product. buffered saline (PBS) prior to EFC or primary and/or secondary antibodies to Western blot analysis if necessary. nonspecific proteins and to the blotting EFC blotting has several advan- membrane itself. Third, the EFC blot REFERENCES tages over Western blotting. First, has the potential of identifying modifi- 1. Kurien, B.T. and R.H. Scofield. 2003. EFC blotting does not require a cations that alter the apparent molecular Protein blotting: a review. J. Immunol. specific antibody and is therefore mass of the protein, such as phosphory- Methods 274:1-15. lation, ubiquitination, and glyco- 2. McIlhinney, R.A. 2004. Generation and use sylation. While antibodies can of epitope-tagged receptors. Methods Mol. �������������� be used to measure such events, Biol. 259:81-98. ������������������������� 3. Patton, W.F. 2002. Detection technologies in they could be sensitive to epitope analysis. J. Chromatogr. B Analyt. modification. Fourth, EFC Technol. Biomed. Life Sci. 771:3-31. blotting provides a significant 4. Eglen, R.M. 2002. Enzyme fragment com- reduction in the amount of time plementation: a flexible high throughput screening assay technology. Assay Drug Dev. and number of steps required to Technol. 1:97-104. detect proteins following transfer 5. Zhao, X., I. Vainshtein, R. Gellibolian, Y. from the gel to the blot. In the Shu, H. Dotimas, X.M. Wang, P. Fung, J. comparison above (Figure 2), the Horecka, et al. 2003. Homogeneous assays for cellular protein degradation using beta- EFC blot was completed within galactosidase complementation: NF-kappaB/ 1.5 h and required only two IkappaB pathway signaling. Assay Drug Dev. reagent additions and one wash Technol. 1:823-833. step. In contrast, the Western blot 6. Vainshtein, I., S. Silveria, P. Kaul, R. required 4 h with multiple reagent Rouhani, R.M. Eglen, and J. Wang. 2002. A high-throughput, nonisotopic, competitive additions and wash steps. The binding assay for kinases using nonselec- main limitation of the EFC blot tive inhibitor probes (ED-NSIP). J. Biomol. is that it cannot be used to detect Screen. 7:507-514. endogenous proteins in cells and 7. Naqvi, T., A. Lim, R. Rouhani, R. Singh, Figure 2. Comparison of enzyme fragment complemen- and R.M. Eglen. 2004. Beta galactosidase tissue. In certain situations, such enzyme fragment complementation as a high- tation (EFC) and Western blot detection sensitivities. as when loading controls can be The nuclear factor of activated T cells protein tagged at its throughput screening protease technology. J. C terminus with ProLabel (NFAT-PL) cell lysate was seri- performed simultaneously with Biomol. Screen. 9:398-408. ally diluted in lysis buffer, separated by sodium dodecyl target detection in Western blot 8. Macian, F. 2005. NFAT proteins: key regula- sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analysis, an EFC blot would tors of T-cell development and function. Nat. and transferred to a single nitrocellulose membrane as Rev. Immunol. 5:472-484. be less efficient. For routine 9. Horsley, V. and G.K. Pavlath. 2002. NFAT: described in Figure 1. The blot, containing duplicate sam- analysis, however, EFC blotting ples sets, was divided in two and probed separately for PL ubiquitous regulator of cell differentiation and NFAT using EFC or Western methods, respectively. offers higher throughput and a and adaptation. J. Cell Biol. 156:771-774. EFC analysis was performed as described in Figure 1. simpler protocol without compro- For Western analysis, the blot was rinsed with water for 2 mising sensitivity. The approach Address correspondence to Neil min, and nonspecific sites were blocked by incubation for can be applied to the detection of W. Charter, DiscoveRx Corporation, 1 h with SuperBlock® (Pierce Biotechnology, Rockford, IL, USA) containing 0.05% Tween® 20. The blot was PL-tagged proteins from whole 42501 Albrae St., Fremont, CA 94538, then incubated for 1 h with mouse anti-NFAT monoclo- cell lysates as well as affinity USA. e-mail: [email protected] nal antibody (Cell Signaling Technology, Danvers, MA, purified and immunoprecipitated USA) diluted 1:1000 in blocking buffer, after which it samples. Finally, protein surveil- To purchase reprints was washed extensively (six times for 10 min each) in lance using PL and EFC offers a phosphate-buffered saline (PBS) with 0.05% Tween 20. of this article, contact The blot was then incubated for 1 h with a goat anti-mouse unique and powerful advantage immunoglobulin G (IgG)-horseradish peroxidase (HRP) over antibody-based methods that [email protected] conjugate (Pierce Biotechnology) diluted 1:1000 in block- extends beyond protein detection ing buffer, followed by further washing. Five milliliters on blots, namely the ability to SuperSignal® West Dura substrate (Pierce Biotechnology) were added to the blot and incubated for 5 min. Signal use the EFC homogenous assay detection was as described in Figure 1. system to rapidly quantify recom-

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