Alexa fluor 568 protein labeling kit

Endocytosis fitness was studied by performing an AF-568-transferrin uptake assays on live T. brucei Lister 427 VSG121 p4716 (GFP-GPI) cells. Bovine transferrin (Thermo Scientific #11107018) was first 2× dialysed in 1× PBS, and then fluorescently labeled with AF-568 using an Alexa Fluor™ 568 Protein Labeling Kit (Thermo Scientific #A10238), following the manufacturer’s instructions. Cells were incubated for 12 h with 20 µg/mL NNV. At 0, 0.5, 1.5, and 6 h, 1 mL samples were taken, accounting for 3 × 10⁵ cells. Each sample was spun down at 7,000 rpm for 2 min, washed in fresh HMI-9 media without serum, and resuspended in 100 µL + 1% BSA + 2 µL 2 mM FMK24 (Mu-Phe-hPhe-FMK, Sigma #M4070), a lysosomal protease inhibitor. The sample was incubated at room temperature for 5 min. Then, 80 nM AF-568-transferrin was added to the sample and incubated at 37 ºC for 1 h. Last, the sample was centrifuged at 7,000 rpm for 2 min, and the cell pellet was resuspended in 20 µL HMI-9 without serum + 4% formaldehyde + 1/10,000 Hoechst 33342. The sample was incubated at room temperature for 10 min and then visualised by fluorescence microscopy.

Cloning, overexpression, and purification of MakA have been previously reported (Dongre et al., 2018 (link)). Alexa Fluor568 labeling of MakA was performed using an Alexa Fluor568 protein labeling kit (Thermo Fisher) according to the manufacturer’s instructions.

All chemicals and reagents were purchased from Sigma (St Louis, MO) unless otherwise stated. The BB94, Purvalanol A, FAK inhibitor II and PP2 compounds were all purchased from EMD BioSciences (La Jolla, CA). All drugs were soluble in DMSO, which was used as a control for all experiments. The BB94 and FAK inhibitor II were used at a final concentration of 5 µM, while Purvalanol A was used at 2 µM and PP2 was used at 10 µM. The Alexa Fluor 568 protein labeling kit used to label the gelatin matrix was purchased from Invitrogen (Eugene, OR), as were all cell culture reagents. Fugene 6 used for transfections was purchased from Roche Diagnostics (Indianapolis, IN). For dynamic visualization of actin, the LifeAct peptide (Riedl et al., 2008 (link)) was cloned into the previously described pLL 5.0 GFP Lenti-viral base vector (Wang & McNiven, 2012 (link)).

Alexa Fluor 568 Protein Labeling Kit (A10238, Invitrogen) was used to label lecithinase for immunofluorescence studies. Briefly, the concentration of lecithinase was determined using the Pierce BCA Protein Assay Kit (23225, ThermoFisher Scientific), and an aliquot of lecithinase was diluted to 2 mg/ml in 500 μl ddH₂O 0.1 M bicarbonate (50 μl) was added to give an optimal pH (~ 8.3) for labeling. This 550 μl reaction volume was added to a vial of Alexa Fluor 568 reactive dye and incubated at room temperature for 1 h with gentle rotation. The reactive dye contains a succinimidyl ester moiety that reacts with primary amines of proteins, producing stable protein–dye conjugates. Excess dye was separated from labeled protein by gel filtration using a Bio‐Rad BioGel P‐30 Fine size exclusion resin. The column was assembled by filling a supplied plastic column with resin until ~ 3 cm from the top and was equilibrated with PBS to ensure proper flow. Next, the labeled protein mixture was added to the column and allowed to settle into the resin, before elution of the protein with elution buffer (10 mM potassium phosphate, 150 mM NaCl, 0.2 mM sodium azide, pH 7.2). The faster running purple band containing labeled lecithinase was collected in 1 ml fractions, whilst the slower running, unincorporated dye was not collected.