Thin-Layer Chromatography in Structure and Recognition Studies of Shiga Toxin Glycosphingolipid Receptors

Primary Human Renal Proximal Tubular Epithelial Cells (pHRPTEpiCs): Shiga Toxin (Stx) Glycosphingolipid Receptors, Stx Susceptibility, and Interaction with Membrane Microdomains

“…Polyclonal chicken IgY anti-Gb3Cer and anti-Gb4Cer antibodies were used for overlay detection of TLC-separated total GSLs derived from pHRPTEpiCs and sucrose gradients fractions (see above). Details can be drawn from numerous previous publications [ 13 , 17 , 77 ] and a very recent dissemination that contains protocols explaining all the laboratory details and trickery practical handling of GSLs and their detection with antibodies and lectins exemplified by various Stx subtypes [ 91 ].…”

Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx

“…For details, refer to previously published protocols [ 28 , 29 , 39 , 67 , 91 , 92 , 94 ]. The laboratory details including the sometimes trickery handling of the amphipathic GSLs for application of the TLC overlay technique using anti-GSL antibodies as well as GSL-specific Stx and the appropriate anti-Stx antibodies have been recently described in great depth by us [ 97 ]. Alkaline phosphatase (AP)-linked rabbit anti-chicken IgY and AP-linked goat anti-mouse IgG antibodies (Code 303-055-033 and Code 115-055-003, both from Dianova, Hamburg, Germany) were used as secondary antibodies.…”

“…TLC overlay immunodetection was performed using anti-Gb3Cer and anti-Gb4Cer antibodies as well as Stx-subtypes Stx1a, Stx2a and Stx2e together with the corresponding anti-Stx1 or anti-Stx2 antibody as previously published [ 67 , 100 , 101 ]. Details of the overlay procedure can be taken from a very recent publication containing protocols that explain all the methodological details and trickery practical handling of GSLs using anti-GSL antibodies and the Stx1a, Stx2a and Stx2e subtypes [ 97 ]. In short, overlay assays were performed with 1:2000 diluted primary chicken IgY anti-Gb3Cer and anti-Gb4Cer antibodies or with solutions of affinity-purified Stx1a, Stx2a, or Stx2e (each 0.33 µg/mL).…”

Ingenious Action of Vibrio cholerae Neuraminidase Recruiting Additional GM1 Cholera Toxin Receptors for Primary Human Colon Epithelial Cells

“…1.05633.0001; Merck, Darmstadt, Germany) were used. Gangliosides were administered to the silica gel surface with a semi-automatic sample applicator (Linomat 5, CAMAG, Muttenz, Switzerland), separated in chloroform/methanol/water (120/85/20, v/v/v) supplemented with 2 mM CaCl 2 and stained with 0.2% orcinol (w/v) in sulfuric acid/deionized water (3/1, v/v) [28]. For the TLC overlay assay with choleragenoid [26,27], the silica gel layer of the plates with separated gangliosides was fixed with polyisobutylmethacrylate (Plexigum P28, Röhm, Darmstadt, Germany).…”

“…BCIP was used with 0.05% (w/v) dissolved in glycine solution (pH 10.4) as the substrate for AP of the secondary antibody that generates an insoluble blue precipitate indicating ganglioside-bound choleragenoid on the silica gel layer of the HPTLC plate. Basics of the TLC overlay procedure have been published recently, providing all of the methodological details, especially exact descriptions of trickery practical handling, for example, of glycosphingolipid separation and silica gel fixation, exemplified with the Shiga toxin, which is an AB 5 toxin comparable to the CT used in this study [28].…”