PRODUCT APPLICATION FOCUS Antibody-free method for protein 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 assay (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 proteins 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 Western blot 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 gel electrophoresis 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.