Bca protein estimation kit

Cells were grown to 80–90% confluency and harvested, and cellular proteins were extracted with lysis buffer [40 mM HEPES-NaOH (pH 7.4), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS,150 mM NaCl] containing 1 X cocktail of protease inhibitors (Roche, Indianapolis, IN). Protein was estimated by BCA protein estimation kit (ThermoFisher Scientific) separated on a 12.5% SDS-polyacrylamide gel and proteins electroblotted to nitrocellulose membranes (Bio-Rad, CA). After blocking with 5% nonfat dry milk and 0.1% Tween 20 in Tris-buffered saline, membranes were incubated at 4°C for 16 h with caveolin-1 anti-mouse polyclonal antibodies (1:5000) (Cell Signaling, CA). The membranes then incubated with peroxidase-labeled secondary antibodies and (Amersham Pharmacia, Piscataway, NJ) developed by Super Signal chemiluminescence substrate (Pierce, Rockford, IL). Actin protein levels were used as a control for adequacy of equal protein loading.

The protein concentration in the samples was measured using the BCA protein estimation kit (Thermo Scientific, Waltham, MA, USA). The BSA standard (10–1000 μg/mL) was used to calculate protein concentration in the unknown samples.

Protein extracts were prepared by lysing the cells in RIPA buffer (Invitrogen), which consisted of 50 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 0.5% deoxycholate, 0.1% SDS, and 1.0% NP-40. 1X Protease inhibitor cocktail (Thermo Scientific) was included during cell lysis. Proteins were quantified by BCA Protein Estimation Kit (Thermo Scientific). 100 µg of each protein extract was loaded on 4–20% mini-PROTEAN TGX gels (Biorad). Western blotting was performed following established protocols. The antibodies and dilutions employed for Western blotting are provided in Table S1.

Whole worm excretory-secretory protein (ESP) extracts of A. lumbricoides, kindly donated by Prof William Horsnell, were prepared and supplied by the Division of Immunology, Department of Pathology from the Faculty of Health Sciences at the University of Cape Town, SA. Adult worms were obtained from patients from the Red Cross War Memorial Children’s Hospital (Cape Town, South Africa), and were used to acquire A. lumbricoides excretory proteins. The A. lumbricoides excretory proteins were obtained by keeping the worms alive at 37 °C in Dulbecco modified essential medium with 1% Pen-strep (Thermofisher Scientific, Waltman, MA, USA), and 1% glucose (wt./vol). The media was collected three times a day. Using Amicon ultra concentrator, extract proteins were concentrated and resuspended in 5 mL of phosphate-buffered saline (Merck). All antigens were measured for protein content with a BCA protein estimation kit (Thermofisher Scientific) or by using the Bradford assay previously described [27 (link)] and stored at −80 °C at a standard concentration of 500 µg/mL until further use.

Ixofin3D was cloned in-frame into the pMT-Bip-V5-His tag vector (Invitrogen, CA) using ixofin3D_DESF and ixofin3D_DESR primers listed in Table S1 and recombinant protein expressed and purified using the Drosophila Expression System (Invitrogen, CA) as described earlier [22] (link). Recombinant protein purity was assessed by SDS-PAGE, and quantified using the BCA protein estimation kit (Thermoscientific, IL).