Secondary goat anti mouse igg hrp

Enzyme-linked immunosorbent assay (ELISA): Goat anti-mouse IgG coated 96-well ELISA microplates (Pierce Biotechnology, Rockford, IL) were rinsed with PBS + 0.05% Tween-20 (PBST). Mouse anti-MUC1-N/C antibody standards and AuNR supernatant samples containing unknown quantities of MUC1-N or -C (100 μL/well) were loaded and allowed to capture for 1 hr at room temperature with gentle mixing. Plates were washed 3X in 200 μL PBST. Secondary goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, 100 uL/well, 1:6000 dilution) was incubated for 1 hour at 37°C with mixing. The plate was washed 3X with PBST and a 1 mg/mL 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfuric acid) (ABTS) solution was prepared in 50 mM citric acid and 100 mM dibasic sodium phosphate. Immediately prior to use, the ABTS solution was mixed with 10 μL 30% H₂O₂ and added to wells (100 μL). ABTS color development based on the presence of HRP-tagged secondary antibody was measured at 405 nm within 15 minutes on a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT). The quantity of AuNR-bound MUC1-N and MUC1-C antibodies was determined by subtracting the concentrations detected in the sample supernatants from the known loading conditions during preparation. Concentrations were calculated from point-to-point linear regression standard curves.

[LOOSESTCells were lysed using NP40 cell lysis buffer (Life Technologies). Cell lysates were separated by SDS-PAGE using a precast 4–15% gel (Bio-Rad, Hercules, CA) and blotted on nitrocellulose membranes (Bio-Rad, Hercules, CA) that were then washed with Tris-buffered saline (TBS) and blocked with fat-free milk in the same buffer for 1 hour at room temperature. The membranes were subject to incubation with a primary rat monoclonal antibody directed against mouse CCR6 (clone CKR-6 (13Q7), dilution 1∶200) or a mouse anti-β-actin antibody (clone ACTBD11B7, dilution 1∶1000) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C. The following day, the membranes were washed and incubated with goat anti-rat IgG-HRP conjugated secondary antibody (for anti-CCR6 primary antibody) and secondary goat anti-mouse IgG-HRP (for anti-β-actin primary antibody) (both at 1∶5000 dilution, Santa Cruz Biotechnology, Inc.). Finally, the membranes were developed by using the Amersham ECL Plus kit (GE Healthcare, Piscataway, NJ) as per the manufacturer's protocol.

Proteins were run on 4–12% SDS polyacrylamide gels, and subsequently transferred to PVDF membranes at 100 V for one hour. All blocking steps were carried out shaking at 30 rpm using 3% fat-free skimmed milk. Primary monoclonal anti-FLAG M2 antibody (Sigma) was used at a concentration of 1:1000. Secondary goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) was used at a concentration of 1:5000. Primary and secondary antibodies were diluted in 3% fat-free skimmed milk. Washing steps were carried out with PBS containing 0.05% Tween 20, and bands were detected using Super Signal West Pico chemiluminescent substrate (Thermo Fisher Scientific).

HEK293T cells (DSMZ no.: ACC 635) were transfected with pcDNA3.1 His-Xpress-ZBTB7A (wild type or mutant). After 24 h, protein was extracted using lysis buffer (50 mM Tris HCl, pH 8.5, 150 mM NaCl, 1% Triton X-100, cOmplete Protease Inhibitor Cocktail). For each reaction, 20 μl protein lysate was incubated in binding buffer (PBS supplemented with 150 mM NaCl resulting in a total salt concentration of nearly 300 mM, 0.1% NP40, 1 mM ETDA) with 10 pM biotinylated double-stranded oligonucleotides that contain either the ZBTB7A consensus binding motif (POK WT; 5′-GGTTAAAAGACCCCTCCCCGAATTCGGATC-3′) or a mutant thereof (POK mut; 5′-GGTTAAAATTTTTCTCCCCGAATTCGGATC-3′). After 1 h of incubation at 4 °C, 10 μl streptavidin agarose beads (Sigma Aldrich) was added to each reaction and incubated for 30 min at 4 °C. Beads were washed three times with binding buffer and resupended in 10 μl Laemmli buffer for subsequent western blot analysis. ZBTB7A protein was detected using an antibody against the Xpress tag (1:5,000 dilution, clone R910-25; Life Technologies) and secondary goat anti-mouse IgG-HRP (1:10,000 dilution, clone sc-2060; Santa Cruz). The uncropped western blot scan underlying Fig. 2c is shown in Supplementary Fig. 9.