Protein samples were run on an SDS-PAGE gel alongside a pre-stained ladder (Invitrogen, Waltham, U.S.) and then transferred onto a nitrocellulose membrane (Bio Rad Laboratories, Hercules, U.S.) in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% (v/v) methanol, pH 8.3) using a TE77 semi-dry transfer unit (Hoefer, Holliston, U.S.) at 200 V, 50 mA/gel for 1 h. The membrane was incubated by blocking solution (TBS-T plus 5% (w/v) skim milk powder) on a shaker at RT for at least 1 h to block nonspecific binding sites of the membrane. Following this step, the membrane was washed twice with TBS-T (20 mM Tris-Cl, 150 mM NaCl, pH 7.6, 0.1% (v/v) Tween-20) for 5 min. The membrane was probed with human sera from patients with invasive GAS infections and healthy donors at 1/100 dilution in probing solution (TBS-T plus 2.5% (w/v) skim milk powder) for 1 h at RT. The membrane was then washed three times with TBS-T for 5 min each and incubated with HRP-conjugated goat anti-human IgG (Abcam) in a probing buffer and incubated for 1 h followed by washing with TBS-T three times for 5 min. Western blots were developed with ECL detection reagent (Amersham Biosciences) and analyzed using a ChemiDocTM imaging system (BioRad).
Hrp conjugated goat anti human igg
Manufactured by Abcam
Sourced in United Kingdom
HRP-conjugated goat anti-human IgG is a secondary antibody used to detect and quantify human immunoglobulin G (IgG) in various immunoassays. The antibody is conjugated with horseradish peroxidase (HRP), which enables colorimetric or chemiluminescent detection.
Automatically generated - may contain errors
Lab products found in correlation
High binding plates, by Corning (2 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Te77 semi dry transfer unit, by Hoefer (1 mentions)
Ecl detection reagent, by Cytiva (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Recombinant human tyro3 protein, by Abnova (1 mentions)
Goat anti human igm, by Abcam (1 mentions)
6 protocols using hrp conjugated goat anti human igg
1
Western Blot Analysis of GAS Infection
2
Recombinant Tyro3 Protein ELISA
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Recombinant human Tyro3 protein (Abnova, Taiwan) was prepared by a wheat germ expression system. The protein was fused with a GST-tag at N-terminal and purified by glutathione sepharose 4 fast flow. Antibody against human Tyro3 receptor in the sera of SLE patients and HCs was determined by an enzyme-linked immunosorbent assay (ELISA) at 450 nm. Ninety-six-well high-binding plates (Corning, New York, USA) were coated with Recombinant human Tyro3 protein overnight at 4°C. The antigen-coated wells were washed three times with PBST buffer (PBS plus 0.05% Tween-20) and blocked with PBST buffer containing 5% bovine serum albumin (BSA) for 2 h at 37°C. The human Tyro3/Dtk antibody (R&D Systems, USA) was used as a positive control. The blocking buffer was removed, and the plates were washed as described above before the addition of 100 μl of serum sample (1 : 100 diluted in 1% BSA). The human sera were incubated for 2 h at room temperature followed by incubation with HRP-conjugated goat anti-human IgG (Abcam, Cambridge, UK) for another 1 h at room temperature. Then, the plates were washed, and 100 μl of tetramethylbenzidine substrate solution was added. It was then stopped by the addition of 50 μl of 0.5 M H2SO4. The absorbance was measured at a wavelength of 450 nm in a microplate reader (Bio-Rad Laboratories, Richmond, USA).
3
Antibody Isoforms Detection by ELISA
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Two antibody isoforms (IgG and IgM) in human serum were assessed using a commercially available BLV indirect gp51 ELISA kit (JNC, Tokyo, Japan), according to the manufacturer’s instructions. Human serum specimens, diluted in 1:50 with the sample dilution buffer in the kit, were used as primary antibodies, followed by HRP-conjugated goat anti-human IgG (diluted as 1/1000) (Abcam PIC.) or goat anti-human IgM (diluted as 1/10,000) (Abcam PIC.) the secondary antibody. All samples were run triplicate. During each assay, to check the validity of the experiment procedure, we used two controls, as indicated in ELISA for anti-BLV p24 antibody.
4
Immunoassay Reagent Preparation
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Phosphate buffered saline (PBS) tablets (Cat. No. P4417), polyoxyethylene sorbitan monolaurate (Tween20) (Cat. P7949), skim Milk powder (Cat. 70166), TMB liquid substrate for ELISA (Cat. No. T0440), sulfuric acid (Cat. No. 84716) and oxalic acid (Cat. No. 241172) were purchased from Sigma-Aldrich. Glassfiber (GFB-R4), polyester (PT-R5) and cellulose (AP080) were purchased from MDI membrane technologies. Nitrocellulose pore size 0.45 µm (1620115) and PVDF pore size 0.2 µm (1620177) were purchased from Bio-Rad. HRP Conjugated goat anti human IgG (Cat. ab97175) was purchased from Abcam. Recombinant protein G (Cat. 21193) and ABTS single solution ready for use (Cat. No. 00-2024) were purchased from Thermo Fisher. Milli-Q ultrafiltered (UF) H2O (with a resistivity of 18.2 MΩ cm at 25 °C) were used in the preparation of all solutions.
5
ELISA for Serum IgG Quantification
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96-well microtiter plates were coated overnight at 4°C with recombinant proteins (Proteintech Group Inc, Chicago, IL, USA) at a final concentration of 1.0 μg/ml. Human serum (1:80 diluted in 1% BSA/PBS) was incubated for 2 h at 37°C. HRP-conjugated goat anti-human IgG (1:4000 diluted) (Abcam Inc, Cambridge, MA, USA) and tetramethylbenzidine (TMB) were used as detecting reagents. The optical density (OD) value at a wavelength of 450 nm was applied.
6
Tyro3 Antibody Detection in SLE
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Recombinant human Tyro3 (Abnova, Taiwan) was prepared by wheat germ expression system. The protein was fused with a GST-tag at N-terminal and puri ed by glutathione sepharose 4 fast ow. Antibody against human Tyro3 receptor in the sera of SLE patients and HCs was determined by an enzyme-linked immunosorbent assay (ELISA). Ninety-six-well high binding plates (Corning, New York, USA) were coated with recombinant human Tyro3 protein in 0.05 mol/L carbonate buffer sodium (pH=9.6) overnight at 4 °C. The antigen-coated wells were washed three times with PBST (PBS plus 0.05% Tween-20) and blocked with PBST containing 5% bovine serum albumin (BSA) for 2 h at 37 °C. The blocking buffer was removed, and the plates were washed as described above before the addition of 100 μl of serum sample (1:100 diluted in 1% BSA). The human sera were incubated for 2 h at room temperature followed by incubation with HRP-conjugated goat anti-human IgG (Abcam, Cambridge, UK) for another 1 h at room temperature. Then, the plates were washed, and 100 μl of tetramethylbenzidine substrate solution was added. The color development was stopped by the addition of 50 μl of 0.5 M H 2 SO 4 . The absorbance was measured at a wavelength of 450 nm in a microplate reader (Bio-Rad Laboratories, Richmond, USA).
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