Tmb solution for western blotting

Immunostaining of glycolipids on TLC sheets was performed according to the method described by Watarai et al. [23 (link)], with certain modifications. Glycolipids (1 μg) were spotted onto TLC silica gel polyester sheets (100 × 100 mm, Polygram, Macherey-Nagel, Germany) and developed with chloroform/methanol/water/acetic acid (60:40:6:0.2 vol.) at room temperature for 30 min and then air-dried. Glycolipids on the TLC sheets were visualized with a sprayed 5-methylresorcinol (Orcinol, Tokyo Chemical Industry, Tokyo, Japan) solution (2 mg/mL in 2 M H₂SO₄) and heated at 110°C for 5 min. For immunostaining, TLC sheets were immersed in PBS containing 1% gelatin, 1% polyvinylpyrrolidone (PVP-K30, Nacalai Tesque, Kyoto, Japan), and 1 mM EDTA, followed by immersion in anti-ASGM1 rabbit mAbs (50 ng/mL) or PoAb (1:1,000) solution and allowed to react for 1 h at room temperature. After washing thrice with T-PBS, HRP-conjugated anti-rabbit IgG (1:40,000; The Jackson Laboratory, Bar Harbor, ME, USA) was added. After washing thrice with T-PBS, the TLC sheets were incubated in TMB solution for western blotting (Nacalai Tesque, Kyoto, Japan). Consequently, the bound antibody was visualized in blue.

Western blotting analysis was performed by using a horseradish peroxidase-conjugated anti-His-tag antibody (Anti-His-tag mAb-HPR-DirecT, MBL, Nagoya, Japan). Samples were subjected to SDS-PAGE, followed by electroblotting to a polyvinylidene difluoride membrane (GE Healthcare). For signal detection, Blocking One (Nacalai Tesque, Kyoto, Japan) and TMB solution for Western blotting (Nacalai Tesque, Kyoto, Japan) were employed.