Streptavidin hrp

Hemopexin was measured in a sandwich ELISA. A microtiter plate (Greiner Bio-One™) was coated with 500 ng/ml anti-human hemopexin capture antibody (Bioporto) and incubated at 4°C overnight. Subsequently, standard dilutions of hemopexin from human plasma (0–50 ng/ml, Sigma-Aldrich) as well as diluted patient samples were added to the plate. Next 100 ng/ml of the detection antibody biotinylated anti-human hemopexin (Bioporto) was added to the plate followed by 1:1,000 dilution of streptavidin-HRP (RPN1231V GE healthcare). Subsequently, TMB Substrate (Sigma-Aldrich) was added and the absorbance was measured at wavelength of 450 nanometer (nm) with Victor 3 V multilabel plate reader (PerkinElmer).
Plasma HO-1 levels were determined by a sandwich ELISA using the human HO-1 matched antibody pair kit (Abcam) according to the manufacturer’s instructions.
Hemolysis and icterus index (respectively H-index and I-index) were determined using Serum Index Gen. 2 assay according manufacturer’s instructions (Cobas c system, Roche Diagnostics). H-index was used as marker for free hemoglobin. I-index was used as marker for bilirubin (both conjugated and unconjugated). H-index and I-index were determined based on absorbance measurements (570 and 480 nm primary wavelength and 700 and 505 nm secondary wavelength respectively) and expressed as µmol/L.

To analyze XPA–DNA interactions, a loop‐containing oligonucleotide was used as a XPA binding substrate as described previously 52 with modifications. Briefly, recombinant XPA was pre‐incubated for 30 min with PAR (of defined chain length and concentration as indicated) in EMSA binding buffer (25 mm Hepes‐KOH, pH 8.3; 30 mm KCl; 0.4 mm MgCl₂; 1 mm EDTA; 10% glycerol; 45 μg·mL⁻¹ BSA; 0.9 mm dithiothreitol) before adding 20 nm of the biotinylated duplex oligonucleotide. Samples were incubated for 30 min and then subjected to Tris/borate/EDTA‐PAGE for separation of free and XPA‐bound DNA. The gel was semidry blotted onto a nylon membrane, DNA was immobilized by incubating the membrane for 1.5 h at 95 °C. Unspecific binding sites were blocked by incubating the membrane in TBST supplemented with 5% (w/v) skim milk. Then, the membrane was washed three times in TBST, incubated in streptavidin‐HRP (dilution 1 : 2500; GE Healthcare), washed again and chemiluminescence was analyzed. Quantitative analysis of the band shift was performed using imagej. Relative band shift was calculated by dividing the XPA‐bound‐DNA signal intensity by the overall signal intensity of the whole lane followed by normalization to control samples with XPA/DNA only.

Cells were lysed in 1% triton lysis buffer. For untagged LRP6 protein, IP was performed with mouse monoclonal anti-LRP6 (Abcam, Cambridge, UK) and protein G agarose (Thermo Fisher Scientific, Idstein, Germany). For flag-tagged LRP6 protein, IP was performed using anti-Flag M2 affinity gel (Sigma-Aldrich, Taufkirchen, Germany). IP products were used for lectin blotting. The PVDF membrane (Bio-Rad Laboratories, Feldkirchen, Germany) was blocked with RIPA buffer (50 mM Tris-HCL, 0.1% Trito X-100, 0.1% Sodium Deoxycholate, 150 mM NaCl, 0.1% SDS, adjusted to pH 7.4) for 1 h at room temperature. Amounts of 2 μg/mL biotinylated Lycopersicon esculentum lectin (LEL, Vector Laboratories) or 5 μg/mL biotinylated Concanavalin A (ConA) (Vector Laboratories, Newark, CA, USA) were prepared freshly in RIPA buffer and incubated with the membrane at 4 °C overnight. After washing with RIPA buffer, the membrane was incubated with 1/2500 streptavidin-HRP (GE HealthCare, Braunschweig, Germany) for 2 h at room temperature. The membrane was washed with TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, adjusted to pH 8.0) buffer before subjecting to ECL detection.

To analyze the generation of C3(H₂O) in plasma subjected to repeated F/T cycles, an in-house C3(H₂O) ELISA was used (11 (link)). All washing steps were performed three times with the wash buffer PBS 0.05% Tween 20 and PBS 0.05% Tween20 with 10 mM EDTA and 1% BSA was used as blocking and dilution buffer. All incubations steps were performed at RT on a plate shaker at 700 rpm. In brief, a 96-well Nunc Maxisorp plate (Thermo Fisher Scientific, Denmark) was coated with 100 µL of 0.8 µg/mL mAb anti-C3a 4SD17.3 in PBS overnight at 4°C. The plate was washed and subsequently blocked with 200 µL blocking buffer for 60 min and then was washed again. Plasma samples were diluted 1/200 and added in duplicate to the plate (100 µL/well) and incubated for 60 min. For the standard curve, pooled plasma from all donors were diluted 1/200 and spiked with 0.5-0.03 µg/mL C3 treated in VB⁺⁺, pH 11.0 (prepared as described in Table 1), to represent C3(H₂O). The plate was washed, followed by the addition of 100 µL 1 µg/mL of the biotinylated detection antibody anti-human C3d (Dako) for 60 min. After washing the plate, 100 µL of streptavidin-HRP (GE Healthcare Uppsala, Sweden) diluted 1/500 was added for 15 min. The plate was again washed followed by the addition of 100 µL TMB. Finally, the reaction was stopped using 100 µL 1M H₂SO₄ and the absorbance was measured at 450 nm.

ELISA plates (Costar) were coated with recombinant proteins (10 μg/ml), as indicated in the figure legends, blocked with 1% BSA then incubated with test proteins including FLAG-tagged Clec ectodomain fragments, GST-RNF41 proteins or controls. Binding was detected with anti-FLAG HRP or rabbit anti-RNF41 Ab (Bethyl Laboratories, A300-049A) and anti-rabbit Ig HRP (Bio-Rad). Statistical analysis of ELISA data was performed by an unpaired two-tailed t-test on log-transformed data.
To measure the effect of pH on Clec9A binding to RNF41, ELISA plates were coated with GST-tagged RNF41 proteins (10 μg/ml), control GST (10 μg/ml), or actin complexes (platelet actin pre-incubated with GST-tagged βII-spectrin actin-binding domain (ABD): 10 μg/ml actin; 2.5 μg/ml ABD), blocked then incubated with biotinylated FLAG-tagged recombinant Clec9A-ectodomain proteins and controls in pH 4–8 adjusted blocking buffer. Binding was detected with streptavidin-HRP (GE Healthcare) in pH-adjusted blocking buffer.

Anti-hemagglutinin (HA), anti-phospho-FGFRs (Y653/Y654) were purchased from Cell Signaling Technology. Anti-FGF-2 as well as HRP-goat anti-rabbit conjugate and HRP-goat anti-mouse conjugate were from Santa Cruz. Streptavidin-HRP was purchased from GE Healthcare. Streptavidin, Alexa Fluor 488 conjugate and Dynabeads (R) M-270 Streptavidin were purchased from Invitrogen. IgG-Cy3-goat anti-mouse secondary antibodies were purchased from Chemicon.