The identification of high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation

Antibodies

All sFRP-1 antibodies are listed in Table 2. Peroxidase-conjugated goat anti-rabbit and donkey anti-goat antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and A15999).

Cell culture

A549 WT and SFRP1 KO used are listed in Table 1. Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065), 100 IU penicillin and 100 μg/ml streptomycin (Wisent cat. number 450201). Cells were starved in DMEM high-glucose containing L-glutamate and penicillin/streptomycin.

Antibody screening by Western Blot on culture media

A549 cells (WT and SFRP1 KO) were washed 3× with PBS and starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500 × g to eliminate cells and larger contaminants, then for 10 min at 4500 × g to eliminate smaller contaminants. Culture media were concentrated by centrifuging at 4000 × g for 10 min using Amicon Ultra-15 Centrifugal Filter Units with a membrane NMWL of 10 kDa (MilliporeSigma cat. number UFC901024).

Western Blots were performed as described in our standard operating procedure.⁸ Western Blots were performed with large 8-16% gradient polyacrylamide gels and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin in TBS with 0,1% Tween 20 (TBST). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/ml in TBST with 5% milk for 1 hr at room temperature followed by three more washes with TBST. Membranes were incubated with ECL from Pierce (cat. number 32106) prior to detection with HyBlot CL autoradiography films from Denville (cat. number 1159T41).

Antibody screening by immunoprecipitation on culture media

Immunoprecipitation was performed as described in our standard operating procedure.⁹ Antibody-bead conjugates were prepared by adding 1 μg of antibody to 500 ul of Pierce IP Buffer from Thermo Fisher Scientific (cat. number 87788) in a microcentrifuge tube, together with 30 μl of Dynabeads protein A - (for rabbit antibodies) or protein G - (for goat antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) was supplemented with the Halt Protease Inhibitor Cocktail 100X from Thermo Fisher Scientific (cat. number 78446) at a final concentration of 1×. Tubes were rocked for ~2 hrs at 4°C followed by two washes to remove unbound antibodies.

Starved A549 WT culture media were concentrated as described above. 1 ml aliquots at 0.5 mg/ml of lysate were incubated with an antibody-bead conjugate for ~2 hrs at 4°C. Following centrifugation, the unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of IP Lysis Buffer and processed for SDS-PAGE and Western Blot on 8-16% polyacrylamide gels. Prot-A: HRP (MilliporeSigma, cat. number P8651) was used as a secondary detection system at a dilution of 0.4 μg/ml.