The purification of specific antibodies was performed according to a previous report [22 (link)]. In brief, recombinant human PD-1 was spotted onto a nitrocellulose filter and incubated with the purified IgG from SLE patients. After washing with PBST, the antibodies were eluted with 100 mM of glycine (pH = 2.8). The eluted antibodies were immediately neutralized with 1 M of Tris-HCl (pH = 8.0). Normal human IgG was purchased from Millipore (Darmstadt, Germany). The purified PD-1 antibodies were concentrated with a final concentration of 1 mg/ml. After sterile filtration, the autoantibodies were stored as small aliquot package in -80 °C.
Normal human igg
Manufactured by Merck Group
Sourced in United States
Normal human IgG is a type of immunoglobulin G (IgG) that is naturally present in the human body. IgG is the most abundant antibody isotype found in the blood and plays a crucial role in the adaptive immune response. Normal human IgG is commonly used as a reference standard or control in various laboratory tests and assays.
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Lab products found in correlation
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (3 mentions)
Anti dact1, by Abcam (2 mentions)
Normal mouse igg, by Jackson ImmunoResearch (1 mentions)
Dynasore, by Merck Group (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Cp 724714, by Selleck Chemicals (1 mentions)
Y 1127, by FroggaBio (1 mentions)
Y 1139, by FroggaBio (1 mentions)
Y 1196, by FroggaBio (1 mentions)
Y 1248, by FroggaBio (1 mentions)
Trastuzumab, by Roche (1 mentions)
Expicho s expression system, by Thermo Fisher Scientific (1 mentions)
Human igg, by Fujifilm (1 mentions)
Pcag ble vector, by Fujifilm (1 mentions)
Pcag neo vector, by Fujifilm (1 mentions)
Spri biochip, by Horiba (1 mentions)
Scrubbergen software, by Horiba (1 mentions)
10 protocols using normal human igg
1
Purification of Anti-PD-1 Autoantibodies
2
Analyzing RNA-Protein Interactions with RIP Assay
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RNA immunoprecipitation (RIP) assay was conducted using Magna RIP RNA-Binding
Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the
manufacturer’s instructions. In brief, cells were washed with ice-cold PBS and
then lysed in radioimmunoprecipitation buffer at 4°C for 30 min. The whole cell
lysates were incubated with protein A/G sepharose beads conjugated to antibodies
against Ago2 (Millipore) or normal human IgG (Millipore). Western blotting and
qPCR were used to analyze immunoprecipitated RNA and total RNA from the whole
cell lysates (input controls).
Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the
manufacturer’s instructions. In brief, cells were washed with ice-cold PBS and
then lysed in radioimmunoprecipitation buffer at 4°C for 30 min. The whole cell
lysates were incubated with protein A/G sepharose beads conjugated to antibodies
against Ago2 (Millipore) or normal human IgG (Millipore). Western blotting and
qPCR were used to analyze immunoprecipitated RNA and total RNA from the whole
cell lysates (input controls).
3
DACT1 RNA Immunoprecipitation Assay
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RIP assay was performed by using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore).
In brief, the treated cells were washed by ice-cold PBS and lysed in a radioimmu-noprecipitation buffer for 30 minutes at 4 °C. Next, the whole cell lysate was incubated with A/G sepharose beads coupled with anti-DACT1 (Abcam) or normal human IgG (Millipore) antibodies. The precipitated RNA was analyzed by qPCR.
In brief, the treated cells were washed by ice-cold PBS and lysed in a radioimmu-noprecipitation buffer for 30 minutes at 4 °C. Next, the whole cell lysate was incubated with A/G sepharose beads coupled with anti-DACT1 (Abcam) or normal human IgG (Millipore) antibodies. The precipitated RNA was analyzed by qPCR.
4
DACT1 RNA Immunoprecipitation Assay
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RIP assay was performed by using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore).
In brief, the treated cells were washed by ice-cold PBS and lysed in a radioimmu-noprecipitation buffer for 30 minutes at 4 °C. Next, the whole cell lysate was incubated with A/G sepharose beads coupled with anti-DACT1 (Abcam) or normal human IgG (Millipore) antibodies. The precipitated RNA was analyzed by qPCR.
In brief, the treated cells were washed by ice-cold PBS and lysed in a radioimmu-noprecipitation buffer for 30 minutes at 4 °C. Next, the whole cell lysate was incubated with A/G sepharose beads coupled with anti-DACT1 (Abcam) or normal human IgG (Millipore) antibodies. The precipitated RNA was analyzed by qPCR.
5
PECAM-1 Homophilic Binding Assay
6
Rpgrip1l Knockout Mouse Model and Cell Experiments
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The Rpgrip1l mouse model was described previously [2 (link),4 (link),5 (link),25 (link)]. HaCaT cells were transfected with RPGRIP1L siRNAs (HSC.RNAI.N015272.12.1 and 2, Integrated DNA Technologies, Coralville, IA). Non-targeting (Negative Control) siRNA (NC-1, Integrated DNA Technologies) was used as control siRNA. Twenty-four hours after transfection, cells were switched to high calcium (1.5 mM CaCl2) for designated durations. EGTA was used at 2 mM. Dynasore (Sigma-Aldrich, Saint Louis, MO) and sucrose were used at 50 μM and 400 mM, respectively. PV IgG were purified in Dr. Payne’s laboratory and used at 400 μg/ml. Normal human IgG (Sigma-Aldrich, Saint Louis, MO) was used as control IgG. Method details are provided in the Supplementary Materials and Methods (S1 Text ) online.
7
HER2 Inhibitor Assay Protocol
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HER2 inhibitor CP-724714 was from Selleckchem (Houston, TX, USA). Normal human IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trastuzumab was from Roche (Basel, Switzerland). Mouse monoclonal antibodies to human EGFR (1005), HER2 (A-2) (sc-393712), human EGFR (1005), Akt1/2 (H-136), Erk1/2 (k-23), pT202/Y204 Erk1/2 were from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Rabbit monoclonal antibodies to phospho-Akt (Ser473) and phosphor(Thr308), and rabbit monoclonal antibody to HER2/ErbB2 (29D8) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies to phospho-HER2 Y-1005, Y-1112, Y-1127, Y-1139, Y-1196, and Y-1248 were from FroggaBio (Toronto, ON, Canada). Secondary antibodies used for Western analysis, including Anti-rabbit and anti-mouse RDye® 800 CW and RDye® 650, were from LI-COR biotechnology Inc. (Lincoln, NE, USA). Invitrogen™ Cholera Toxin Subunit B (Recombinant) and Alexa Fluor™ 488 Conjugate were purchased from Fisher Scientific (Ottawa, ON, Canada). All other chemicals were purchased from Sigma-Aldrich (Oakville ON, Canada).
8
Humanization of Anti-PDPN Monoclonal Antibody
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Normal human IgG was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The complementarity determining region (CDR) of LpMab-23 VH, frame sequences of VH in human Ig, and CH of human IgG1 were cloned into the pCAG-Neo vector [FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan] to generate a humanized anti-human PDPN mAb (humLpMab-23). The CDR of LpMab-23 VL, frame sequences of VL in human Ig, and CL of human kappa chain were cloned into the pCAG-Ble vector (Wako). We transfected the antibody expression vectors of humLpMab-23 into ExpiCHO-S or BINDS-09 cells using the ExpiCHO-S Expression System (Thermo Fisher Scientific, Inc.). We named these mAbs humLpMab-23 and humLpMab-23-f, respectively. We purified humLpMab-23 and humLpMab-23-f using Ab-Capcher (ProteNova Co., Ltd., Kagawa, Japan).
9
Sensor Chip Preparation for IgSF-Fc Proteins
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For the preparation of sensor chips, purified IgSF-Fc proteins and normal human IgG (Sigma-Aldrich, St. Louis, MO, USA) in PBS were spotted onto an SPRi-Biochip (HORIBA France) at 10 nL/spot using a dedicated spotter in a 96-spot format at HORIBA Ltd. (Kyoto, Japan). The spotted proteins were immobilized on the chip by amine coupling. The prepared sensor chip was inserted into the instrument according to the manufacturer's instructions.
10
SPRi Analysis of Protein Binding
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Surface plasmon resonance imaging (SPRi) analyses were performed using an SPRi system OpenPlex (HORIBA France, Palaiseau, France) according to a previous report.18 Briefly, 10 µM protein samples and normal human IgG (Sigma‐Aldrich) in PBS were spotted onto an SPRi‐Biochip (HORIBA France) at 10 nl/spot using a DNA Array Spotter (HORIBA Ltd., Kyoto, Japan). The reflectivity of each spot in response to 2.4 µM analyte samples diluted in running buffer (PBS with 0.2% BSA and 0.02% Tween20) was recorded, and the percentage change in reflectivity (%ΔR) was calculated by subtracting the background signal. Data were processed using ScrubberGen software (HORIBA France).
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