Anti mouse igg hrp antibody

Samples of recombinant TbISP (100 ng) or whole cell lysate (5x10⁶ BSF parasites) were separated on a 15% SDS-PAGE gel and transferred to a Hybond C nitrocellulose membrane (GE Healthcare). The membrane was blocked with Tris-buffered saline (TBS) with Tween 20 [25 mM Tris, 150 mM NaCl, 2 mM KCl, 0.1% Tween 20] and 5% w/v milk powder (Marvel) for 1–2 h at room temperature. Anti-TbISP2 was used at 1:500 and incubated for 1–2 h at room temperature. Membranes were washed three times for 5 min each then incubated with an anti-mouse IgG HRP antibody (Promega) at 1:10,000 for 1–2 h at room temperature. The signal was developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce ThermoScientific).
Densitometry was calculated by determining the areas of the detected bands using the ImageJ software (U.S.A National Institutes of Health, Bethesda, MD, USA). The areas were normalized to the respective loading controls by the ratio between sample/loading control. To assess any change between the different samples, the normalized areas of bands for the WT line were considered as 100% (= 1) and the normalized areas of the other lines were then calculated as percentages in relation to the WT.

Cells were grown overnight in 5 ml SC-Ura, 2% raffinose at 30°C. The next day, fresh cultures were started with optical density OD₆₀₀ of 0.5 in 20 ml SC-Ura, 2% galactose at 30° for 6 hrs. Cells were then harvested, frozen in liquid nitrogen, and stored at −80°. For total protein extract, trichloroacetic acid was performed as described previously^{37 (link)} and protein concentration was determined by BCA protein assay kit (Thermo scientific). Protein samples were separated by 4–12% gradient SDS-PAGE (GenScript) and blotted onto nitrocellulose or PVDF membranes. The following primary antibodies were used at 1:5000 dilution: anti-FLAG antibody (GenScript), and anti-GAPDH antibody (Proteintech). Secondary anti-mouse IgG HRP antibody was used at 1:7000 dilution (Promega). ChemiDoc (Bio-Rad) imaging system was used to detect chemiluminescence signals from blots.