Silica gel 60 rp 18 f254s plate

FITC-dextran (150 kDa) was purified using 12 consecutive cycles of ultrafiltration at 8,000 rpm for 10 min (Amicon filters, 100-kDa molecular weight cutoff [MWCO]). The obtained final solution was then analyzed using gel filtration size-exclusion chromatography (Sephadex G-100 19-cm column as the stationary phase and water as the mobile phase) to confirm the absence of any free FITC label. FITC-dextran, 10 kDa and 70 kDa, were purified using a gel filtration size-exclusion chromatography column and the obtained solutions (volume fractions 9 to 10 for the 10-kDa and 7 to 8 for the 70-kDa FITC-dextran) were concentrated using ultrafiltration (Amicon filters, 3 kDa MWCO, one cycle). The obtained concentrated FITC-dextran solutions were then reanalyzed using size-exclusion chromatography to confirm the absence of a free FITC label.
The commercial and purified FITC-dextrans were also analyzed with thin-layer chromatography using silica gel 60 RP-18 F₂₅₄S plate (Merck Life Science). The plates were first developed with 80% methanol and 20% distilled water and dried; the original unpurified FITC-dextrans were fully resolved from their impurities using 20% methanol and 80% PBS pH 7.4. Developed plates were illuminated under a 366-nm ultraviolet lamp (Krackeler Scientific).

DM/CHS- or DM-solubilized protein was purified by gel filtration chromatography equilibrated with a buffer containing 20 mM HEPES–NaOH pH 7.5, 200 mM NaCl and 0.17% (w/v) DM. Protein-bound lipids were extracted in a mixture of chloroform and methanol (1:2, v/v). The lower chloroform phase was collected and dried under a gentle stream of nitrogen gas. The dried lipid film was re-dissolved in 30 μL of chloroform/methanol (1:2, v/v). The extracts were loaded on a TLC Silica gel 60 RP-18 F254s plate (Merck) and developed in a solvent mixture of chloroform/methanol/ammonia/water (5:3:0.3:0.15, v/v/v/v)^{73 (link)}. The plate was dried and stained with potassium permanganate solution (1.5 g of KMnO₄, 10 g of K₂CO₃, and 1.25 mL of 10% NaOH (w/v) in 200 mL H₂O) to visualize lipids.
We also used TLC to determine if the phospholipids used in this study were contaminated with lyso-phospholipids or other impurities. The lyso-phospholipids used as controls were 16:0 Lyso-PG (1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1’-rac-glycerol)), 16:0 Lyso-PC (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine), 16:0 Lyso-PE (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), and 17:1 Lyso-PS (1-(10Z-heptadecenoyl)−2-hydroxy-sn-glycero-3-[phospho-l-serine]) (Avanti Polar Lipids, Inc., purity > 99%; may contain up to 10% 2-Lyso phospholipid isomer).