Startingblock t20 pbs blocking buffer

To determine if mAbs recognise overlapping epitopes on their corresponding antigen, competition-ELISA were performed. 96-well Nunc MaxiSorp plates (Thermo Scientific, Germany) were coated with 500 ng/well of rCgoX or rTPI, respectively, in PBS at 4 °C over night. Afterwards, the plate was blocked with StartingBlock T20 (PBS) Blocking Buffer (Thermo Scientific, Germany) for 60 min at RT. Binding of DyLight-649 conjugated mAbs to recombinant corresponding antigen was analysed in the presence of increasing concentrations of competing unconjugated mAbs. Binding was determined by fluorescence measurement (Ex 646/Em 674) with microplate reader Infinite M1000 (Tecan, Switzerland).

ELISAs against Pfs25 recombinant protein were performed using standardized methodology as previously described for murine (10 (link), 73 (link)) and human (69 (link), 74 (link)) samples, except that plates were blocked with StartingBlock™ T20 (PBS) Blocking Buffer (ThermoFisher Scientific,UK).
For Pfs28 endpoint ELISA, Nunc-Immuno maxisorp plates were coated with recombinant Pfs28 protein, produced from the Drosophila S2 cells. Indicated serum samples were added in duplicates and diluted 3 fold down the plate, followed by the same procedure as for the Pfs25 standardized ELISA. Optical density (OD) was read at 405 nm using an ELx800 absorbance microplate reader (Biotek, UK). The endpoint titer is defined as the X-axis intercept of the dilution curve at an absorbance value ( ± three standard deviations) greater than the OD for a negative control serum sample.

Mosquito carcasses, 30 each, were homogenized by pellet pestle in 100 μl of lysis buffer [42 (link)]. Aliquots of mosquito protein samples were resolved using SDS-polyacrylamide gels (Bio-Rad) and transferred to PVDF membranes (Merck Millipore). Then, membranes were blocked with Starting Block T20 (PBS) Blocking Buffer (Thermo Fisher Scientific) and incubated with the primary antibody against HPX8C (1:3000). Following three washes with TBS containing Tween-20, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated antibody (Sigma, 1:10000). After washing four times with TBS containing Tween-20, membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate reagent for 5 min (Thermo Fisher Scientific) before visualization. The antibody against α-tubulin (Cell Signaling Technology) was used as the loading control.

ELISAs were performed against full-length Pfs25 protein using standardized methodology as previously described (29 (link), 30 (link)). Briefly ELISA plates were coated over-night with Pfs25 protein (2µg/mL, 50 µL per well). The plates were blocked with StartingBlock™ T20 (PBS) Blocking Buffer (ThermoFisher Scientific,UK) and the assay is performed by using a standard curve and internal controls from the reference serum. Unknown test serum samples from immunized volunteers are diluted and added in triplicate to the ELISA plate. After a two hour incubation period, the diluted sera were discarded, the plate was washed and a secondary polyclonal antibody against the γ–chain of human IgG conjugated to alkaline phosphatase (Sigma, UK) was added. After 1 hour incubation, followed by a wash step, the alkaline phosphatase substrate was added. The substrate is left to develop for 25 minutes and the absorbance at 405nm was read using a plate reader. A standard curve and Gen5 ELISA software v3.04 (BioTek, UK) was used to convert the OD405 of individual test samples into arbitrary units (AU). These responses in AU are reported in μg/mL following generation of a conversion factor by calibration-free concentration analysis (CFCA) as described in Supplementary Materials and Methods.

Purified EVs were lysed using radioimmunoprecipitation assay lysis buffer. Two micrograms of EV protein content were separated on a 4%–12% Bis-Tris gel (Thermo Scientific, Rockford, IL) and then blotted onto a PVDF membrane. Nonspecific binding was blocked by Starting Block T20 PBS Blocking Buffer (Thermo Scientific, Rockford, IL) for 1 hour followed by primary antibodies targeting EV membrane protein, CD9 (Abcam, Waltham, MA; 1:10,000), or EV cytoplasmic protein, TSG101 (Abcam, Waltham, MA; 1:10,000), and incubated for 1 hour. Horseradish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Eugene, OR; 1:50,000) was applied to the membrane and incubated for 1 hour. Membranes were washed for 15 minutes three times between incubation periods with PBS + 0.1% Tween 20. Signal was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA) and imaged using an Invitrogen iBright CL1000.

Cells were lysed in 50–100 μl lysis buffer per well of a 6-well plate for 30 min on ice. Lysate was clarified by centrifugation at 14000 rpm for 10 min. A total of 30–40 μg protein for each sample was separated on 4–15% Mini-PROTEAN TGX Stain-Free^™ Precast Gels (Bio-Rad, Hercules, CA) at 200 V for 30 min, and transferred to PVDF membrane using Trans-blot Turbo Transfer System (Bio-Rad, Hercules, CA) at 1.3 A, 25 V for 10 min. Membrane was blocked with StartingBlock T20 (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA) at room temperature for 15 min followed by incubating with human specific anti-FMRP primary antibody (6B8) (BioLegend, San Diego, CA) or anti-eIF4E primary antibody (BD Biosciences, San Jose, CA) at 4°C overnight. The next day, the membrane was washed with blotto buffer (mix 50 ml 10x PBS, 5 g non-fat milk powder, and 1 ml Tween 20 in 450 ml water) for 5 min at room temperature followed by incubation with anti-mouse secondary antibody for 1 h at room temperature. After 3 times of 10 min washes with blotto buffer at room temperature, the membrane was incubated with the clarity western ECL substrate solution (Bio-Rad, Hercules, CA) for 5 min and exposed with the ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA). Image analysis was performed using Image Lab^™ Software (Bio-Rad, Hercules, CA).