4.3.4. High-Performance Thin-Layer Chromatography-Immunostaining (HPTLC-IS)

Specific detection of proteins and peptides were performed using antibodies according to the procedure described by Morschheuser et al. [52]. Following the evaporation of the mobile phase overnight, one half of the HPTLC plate was transferred to a small vessel.

First, the HPTLC plate was incubated two times for 15 min with a buffer containing Tween20 as a blocking reagent. This prevents the primary antibody from binding non-specifically to the plate surface. For preparing the blocking solution, 0.9% sodium chloride, 0.6% tris(hydroxymethyl)aminomethane, and 0.5% Tween20 was used. Subsequently, the blocking buffer was removed and replaced by the primary antibody solution (1:20,000 for intact proteins and 1:12,000 for peptides, both in blocking buffer). The incubation with primary antibodies lasted for 2 h and was followed by three washing steps with the blocking solution. Coating with the secondary antibody was performed for 1 h (diluted 1:2000 in blocking solution). The secondary antibody exhibited specificity against the primary antibody and was HRP-conjugated. After the plates were washed three times with the blocking buffer, pH was lowered by incubation for 1 min with an acidic solution containing 0.06% Tris-HCl (pH 6.0). The staining solution consists of the following substances: 0.06% 3,3′,5,5′-tetramethylbenzidine, 0.2% dioctyl sulfosuccinate sodium salt, 0.7% citric acid monohydrate, 1.8% sodium hydrogen phosphate dihydrate, 25% ethanol. Just before staining, 0.15% hydrogen peroxide was added. The TLC plate was incubated in the staining solution for 10 min until blue bands appeared. Upon oxidation, TMB forms a water-soluble blue reaction product that can be measured spectrophotometrically at 650 nm. All incubations were executed at room temperature and on an orbital shaker at 70 rpm. Finally, the analytes were detected at white light and documented using a photodocumentation system (TLC visualizer, CAMAG AG, Muttenz, Switzerland).