A Guide to Using SDS-PAGE to Characterize Antibody-Oligonucleotide Conjugates

Principle of SDS-PAGE

SDS-PAGE separates proteins primarily based on molecular weight. The detergent sodium dodecyl sulfate (SDS) denatures proteins and coats them with a uniform negative charge proportional to their length, effectively masking their intrinsic charge differences. When an electric field is applied through a polyacrylamide gel, the negatively charged protein-SDS complexes migrate toward the anode. Smaller proteins move faster through the pores of the gel, while larger ones migrate more slowly, resulting in size-based separation.

Application to Antibody-Oligonucleotide Conjugates

Analysis of antibodies and their conjugates can be performed under either reducing or non-reducing conditions. Reducing conditions break disulfide bonds, separating heavy (H) and light (L) chains, while non-reducing conditions preserve disulfide linkages, allowing the antibody to run as an intact antibody H₂L₂ species. The choice between these conditions depends on the size of the attached oligonucleotide and the information sought, as discussed below.

Detecting Successful Conjugation

A 4-12% Bis-Tris gel is well suited for running both reduced and non-reduced samples. When an antibody is conjugated to an oligonucleotide, the resulting increase in molecular mass can often be detected as a band shift, i.e., the conjugate migrates more slowly than the unconjugated control.

The extent of this shift depends on both the size of the oligonucleotide and the polypeptide chain involved. For a given oligo, the magnitude of the shift generally follows the order L > H > IgG. Thus, non-reducing gels are more useful for large oligo-IgG conjugates, whereas reducing gels provide better resolution when short oligos are used.

While reducing gels enhance band separation, they do not allow accurate quantification of residual free antibody, which, if present, may reduce downstream assay sensitivity.

Typically, DNA strands of 20-80 nucleotides are conjugated to antibodies. Multiple conjugation events per antibody may produce a ladder-like pattern, where each successive band corresponds to one additional oligo. Site-specific conjugation methods usually yield a narrower distribution, depending on the number and location of conjugation sites.

Figure 1 shows a non-reducing SDS-PAGE of a large (67-mer) oligo-monoclonal IgG conjugate stained for protein with Coomassie Blue. Compared with the unconjugated antibody (lane 1), the antibody-oligo conjugate (lane 2) displays a clear upward band shift, forming a ladder corresponding to one (1) to four (4) attached oligos. A small amount of free antibody may still be visible. With polyclonal antibodies, discrete ladders are less common, though the presence of free antibody can still be inferred.

Figure 1. Coomassie dye-stained gel, non-reducing. Lane 1, IgG; lane 2, conjugate; lane 3, protein markers.

When much shorter oligos are used, the band shift under non-reducing conditions becomes less pronounced, making it difficult to distinguish conjugated from unconjugated antibody. In such cases, reducing gels provide clearer evidence of successful conjugation. They do not directly indicate the proportion of free antibody because even if all IgG molecules carry at least one oligonucleotide, the unconjugated H and L chains still migrate independently.

Figure 2 shows decreasing oligo incorporation into H and L chains for four reduced 32-mer oligo-monoclonal IgG conjugates across lanes 3 to 6, compared with controls (lanes 2 and 8). Lane 9 corresponds to the lane 3 sample except under non-reducing conditions, but the resolution is insufficient to allow conclusions to be drawn about the pattern of incorporation.

Figure 2. Lane 1, protein markers; lane 2, IgG; lane 3-6, conjugates with decreasing oligo input; lanes 2 & 8, control IgG; lane 9, non-reduced conjugate.

While the absence of free antibody cannot be confirmed from a reducing gel alone, an obvious H+2 conjugate band is generally missing if significant free antibody remains, serving as an indicative, though not definitive, metric of conjugation efficiency. Lanes 3-5 illustrate the far greater band shift after attaching oligo to the L chain. For very short oligos, a ladder can be accommodated within the clear space below the H chain.

Characterizing Site of Conjugation

To determine whether the oligonucleotide is attached to the heavy or light chain, the gel must be run under reducing conditions. In stochastic lysine-based conjugation methods, oligonucleotides are more likely to attach to heavy chains (as illustrated in Figure 2), though a fraction of light chains also exhibit a shift in mobility. In contrast, site-directed methods may result in conjugation exclusively to H or L chains (or to both), depending on the engineered attachment site.

Oligonucleotide-Specific Detection

While SDS-PAGE primarily separates proteins, it also provides information on oligonucleotide stability, since partial detachment of the oligo may lead to a redistribution of protein bands over time.

Direct visualization of the oligonucleotide can be achieved using SYBR Gold staining (performed before protein staining) or by incorporation of a fluorophore into the oligonucleotide during synthesis.

For a more complete characterization of conjugates, SDS-PAGE is often complemented by UV–Vis spectroscopy, for assessing absorbance contributions from both protein and nucleic acid; mass spectrometry, for precise mass determination; and capillary electrophoresis or size-exclusion chromatography (SEC), for evaluating size distribution and aggregation.

Conclusion

SDS-PAGE is an indispensable, low-cost, and straightforward method for characterizing antibody-oligonucleotide conjugates. By comparing reducing and non-reducing gels, monitoring mobility shifts, and applying appropriate staining techniques, it is possible to confirm successful conjugation, estimate efficiency, and evaluate product uniformity. Although complementary analytical techniques are necessary for precise quantification, SDS-PAGE provides an essential visual confirmation within the workflow of AOC development and quality control.