Specific, sensitive and quantitative protein detection by in-gel fluorescence

Abstract

Recombinant proteins in complex solutions are typically detected with tag-specific antibodies in Western blots. Here we describe an antibody-free alternative in which tagged proteins are detected directly in polyacrylamide gels. For this, the highly specific protein ligase Connectase is used to selectively fuse fluorophores to target proteins carrying a recognition sequence, the CnTag. Compared to Western blots, this procedure is faster, more sensitive, offers a better signal-to-noise ratio, requires no optimization for different samples, allows more reproducible and accurate quantifications, and uses freely available reagents. With these advantages, this method represents a promising alternative to the state of the art and may facilitate studies on recombinant proteins.

Fig. 1. Schematic representation of a Connectase-mediated ligation of two substrates, N and C (a), and AlphaFold model (b) of M. mazei Connectase (white) in complex with its recognition sequence (red, cyan).

Connectase forms an amide bond between its active site residue (Thr-1) and the N-terminal part of a substrate (N-Cnt), while the C-terminal part of the substrate is cleaved. The reaction is reversible so that the amide bond in the original substrate (red) can be reconstituted. Alternatively, a new fusion product with an alternative C-terminal fragment (blue) can be formed.

Fig. 2. Schematic representation of the in-gel fluorescence detection method.

Connectase is pre-loaded with a fluorescent peptide substrate (1) and mixed with a sample containing a CnTagged Protein of Interest (2). After a short incubation step, the fluorescent protein of interest can be visualized on a polyacrylamide gel (3).

Fig. 3. Detection of CnTagged proteins in cell extract via in-gel fluorescence and Coomassie stain of the same SDS-gels.

a Small quantities (125 fmol) of CnTagged proteins were mixed with large quantities (~20 µg) of E. coli cell extract and visualized via in-gel fluorescence (left) or with Coomassie (right). The shown proteins are Single domain Antibody (SdAb), Ubiquitin (Ub), GroES, Glutathione-S-Transferase (GST), Ubiquitin-conjugating enzyme (E2), Ubiquitin-activating enzyme (E1), Heat-shock protein 20 kDa (Hsp20), Proteasome subunit Alpha (PsmA), Outer membrane phospholipase A1 (OmpLA) and Cyclophilin A (CyP). The molecular weights and native assembly states of the proteins are indicated. The employed fluorophore-Connectase conjugate (N-Cnt, see control in lane 1) is only visible as a very faint band in presence of CnTagged POI, indicating an effective transfer of the fluorophore. In high-percentage gels, residual fluorescent peptide substrate (~3 kDa) can be seen at the bottom of the gel (see Supplementary Information). Source data are provided as a Source Data file. b Small quantities (125 fmol) of CnTagged E1 protein were mixed with large quantities of Spodoptera frugiperda SF9, human HEK293, Bacillus subtilis, Pseudomonas fluorescens or Sulfolobus solfataricus cell extracts and visualized via in-gel fluorescence (left) or with Coomassie (right). Source data are provided as a Source Data file.

Fig. 4. Signal-to-substrate relationship in in-gel fluorescence assays.

A serial dilution (from right to left) of CnTagged proteins was detected via in-gel fluorescence, and the normalized densitometric data was plotted. For each protein, three independent experiments (red, green, blue; n = 3) were conducted. The band corresponding to residual N-Cnt is shown for one representative experiment. Source data are provided as a Source Data file.

Fig. 5. Sensitivity of Western blot and in-gel fluorescence (IGF) experiments.

a Three CnTagged or cMyc-tagged proteins (Actin, SdAb, Ub) were expressed in HEK293 cells. Lysate samples containing the indicated protein quantities were analyzed via in-gel fluorescence or Western blot. (* Ub was detected as a monomer and in conjugates (see Source Data file)). b Three CnTagged or cMyc-tagged proteins (E1, GST, SdAb) were expressed in E. coli and purified. Samples containing the indicated molar protein quantities were analyzed via in-gel fluorescence or Western blot. In-gel fluorescence bands for POI quantities above 10 fmol are equally intense due to the limited amount of used reagent (~6 fmol N-Cnt; see also Fig. 4). Source data are provided as a Source Data file.

Fig. 6. Protein quantification with the in-gel fluorescence competition assay.

a Two CnTagged proteins, the reference (Ref) and the protein of interest (POI), compete for fluorophore-conjugated Connectase. b–d Three competition experiments (n = 2 replicates colored red and blue) with constant amounts (125 fmol) of a reference protein (SdAb) and increasing quantities of a protein of interest (Ub (a), Hsp20 (b), GroES (c)). The reactions were separated on polyacrylamide gels (top layer), allowing the densitometric quantification of fluorescent protein bands. The resulting data is represented in plots (middle layer; the same plots on a linear scale are shown in Supplementary Fig. 6), revealing a linear relationship between signal ratio and protein of interest quantities in all cases. The individual curves are shifted in Y-direction by the factor k (see Eq (2); k_(Ub/SdAb) = 0.518 (Exp. 1) and 0.509 (Exp. 2; the average of both values is shown in the plot); k_(Hsp20/SdAb) = 1.272 (Exp. 1) and 1.402 (Exp. 2); k_(GroES/SdAb) = 1.024 (Exp. 1) and 1.061 (Exp. 2)). The linear relationship allows the quantification of target proteins in unknown samples (see Fig. 7). The error of such quantifications (i.e., the deviation from Eq. (2) is represented in plots (bottom layer). The most accurate values are obtained at signal ratio 0.1–10 (dotted lines). Source data are provided as a Source Data file.

Fig. 7. Workflow for relative (3A) and absolute (3B) protein quantification.

Although the results are visualized in plots, it is generally sufficient to use two simple equations for relative (Eq. (1)) and absolute (Eq. (2)) protein quantifications.

Fig. 8. Protein quantification with Western blots.

a–c Shown are three Western blot experiments (n = 3 replicates colored red, green, and blue) with increasing quantities of a cMyc-tagged protein of interest (SdAb (a), E1 (b) and Hsp20 (c); top layer). The resulting POI signals were normalized (100% signal at 12,500 fmol) and plotted against the used POI quantities (middle layer; the same plots on a linear scale are shown in Supplementary Fig. 8). The linear range is indicated by dotted lines and data fits (y = mx) to these linear ranges are indicated by solid lines. The deviation between these fits and the actual data is shown in the bottom layer. Source data are provided as a Source Data file.