Proteins were denatured in SDS sample buffer (Bio-Rad) at 95 °C for 5 min, separated by SDS-PAGE on NuPAGE Novex 10%, 12% or 4–12% Bis-Tris gels (Invitrogen) in MOPS or MES buffer and transferred to nitrocellulose membranes (0.45 μm pore size; Bio-Rad). Membranes were blocked in 5% non-fat milk for 1 h and incubated overnight at 4 °C with the primary antibodies at 1:500 for anti-SIRT2 and anti-SNRPE, 1:1000 for anti-BRCA1, anti-SLC2A1, anti-RRM2, anti-CDC6, anti-RAE1, and anti-STC1, 1:2000 for anti-QKI and anti-LIG1, 1:4000 for anti-β-actin, 1:5000 for anti-lamin A/C, 1:8000 for anti RAD51 and 1:10000 for anti-GAPDH and anti-SRSF1. Secondary antibodies (anti-rabbit IgG NA934 or anti-mouse IgG NA931; 1:2000; GE Healthcare) were applied for 1 h at room temperature and chemiluminescent detection was performed using Lumi-Light PLUS (Roche, Manhein, Germany).
Anti mouse igg na931
Manufactured by GE Healthcare
Sourced in Germany
Anti-mouse IgG NA931 is a secondary antibody reagent used in various immunoassay and immunodetection techniques. It is directed against mouse immunoglobulin G (IgG) and is designed for the detection and quantification of mouse IgG-containing samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg na934, by GE Healthcare (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Lumi lightplus, by Roche (1 mentions)
Sds sample buffer, by Bio-Rad (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Opal four color ihc kit, by PerkinElmer (1 mentions)
Nel794001kt, by PerkinElmer (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Envision reagent, by Agilent Technologies (1 mentions)
Anti myc sc 40, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg na934v, by GE Healthcare (1 mentions)
Trans blot turbo transfert pack, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Neun antibody mab377, by Merck Group (1 mentions)
Neurod1, by Cell Signaling Technology (1 mentions)
9 protocols using anti mouse igg na931
1
Optimized Protein Detection Protocol
2
Immunohistochemical Staining of Gastric Cancer
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Paraffin blocks were sectioned and subjected to immunohistochemical staining using the Envision reagent (Dako, K4002). Antigen retrieval was performed by placing sections in Target retrieval solution (Dako, PH6 S1699, PH9 S2367) and heating to 95 °C in a water bath, according to the manufacturer's instructions. For the immunostaining of human gastric cancer specimens, anti‐ST3G5 antibody (Proteintech) was used at dilutions of 1 : 400. In co‐immunostaining experiments, sections were sequentially stained with each antibody using an Opal™ four‐color IHC Kit and fluorescently conjugated tyramide according to the manufacturer's instructions (PerkinElmer, Waltham, MA, USA, NEL794001KT). Horseradish peroxidase (HRP)‐conjugated secondary antibody (GE Healthcare, anti‐mouse IgG: NA931, anti‐rabbit IgG: NA934) was added for 10 min, and incubated with Opal kit working solution including the desired fluorophore. Tissues underwent the microwave treatment for removal of primary and secondary antibodies before another round of staining according to the Opal Multiplex IHC Assay Development Guide and Image Acquisition Information (Akoya Biosciences, Menlo Park, CA, USA).
3
Western Blot Protein Detection
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The general procedure for Western blotting was performed as reported previously [22 (link),23 (link)]. The following antibodies were used: anti-myc (sc-40; 1:1000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA); alpha-tubulin (CP06; 1:1000 dilution, Merck Millipore, Darmstadt, Germany); anti-mouse IgG (NA931; 1:5000 dilution, GE Healthcare, Chicago, IL, USA); and anti-rabbit IgG (NA934; 1:5000 dilution, GE Healthcare, Chicago, IL, USA).
4
Extracellular Vesicle Protein Extraction and Analysis
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Proteins were extracted from HeLa cells derived-lEVs and -sEV in RIPA buffer as previously described [40 (link)] and their concentration was measured using Bradford assay (#5,000,006, Bio-Rad Laboratories) according to the manufacturer instructions. Ten µg of proteins were separated on 4–12% SDS-PAGE and transferred on 0.22 µm nitrocellulose membranes (Trans-Blot® Turbo™ Transfert Pack, Bio-rad). After blocking with 5% non-fat dry milk in TBS-Tween 0.1% buffer for 1 h at room temperature, the membranes were incubated with primary antibodies at a dilution 1:1000 in TBS-Tween 0.1% buffer with 5% non-fat dry milk (Tetraspanin CD81: sc-166029, Santa Cruz Biotechnology; MVP (major volt protein): 16,478-1-AP, Proteintech) or with 5% BSA (Tetraspanin CD9: #13,403, Cell Signalling) at 4 °C with gentle shaking overnight. The membranes were then washed three times for 10 min with TBS-Tween 0.1% buffer and incubated for 1 h with the corresponding horseradish peroxidase-labelled secondary antibodies (anti-rabbit IgG NA934V and anti-mouse IgG NA931, GE healthcare) at a dilution 1:5000 in the blocking solution. After three washes with TBS-Tween 0.1% buffer, the membranes were incubated for 5 min with Clarity™ Western ECL Substrate (Bio-Rad Laboratories). Images were acquired using ChemiDoc Imaging System (Bio-Rad Laboratories).
5
Isolation and Western Blotting of Cilia
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Cilia were isolated by a previously reported method (43 (link)), demembranated with 0.1% Igepal-CA630 (I3021; Sigma-Aldrich), and subjected to Western blotting. The anti-ODA-IC2 antibody (D6168; Sigma Aldrich) was used as a primary antibody, and anti-mouse IgG (NA931; GE Health Care) was used as a secondary antibody.
6
Antibody Panel for Neurogenesis Analysis
7
Western Blot Protocol for Protein Detection
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Cell lysis buffer (typically RIPA buffer) was used to lyse the cells on ice, and the protein supernatant collected following centrifugation. Protein concentrations were calculated using the Pierce BCA protein assay kit (Thermo Scientific)0.20 μg of heat denatured protein was loaded and run on pre-cast linear gradient gels (Bio-rad 161-1123EDU) A Trans-Blot Turbo transfer system (Bio-Rad) was used to transfer proteins to a PVDF membrane (Bio-rad 1704156EDU), which were blocked in 5% milk powder in PBS (w/v) for 1 h at RT and then probed with primary antibody diluted in 1% milk powder in PBS overnight at 4 °C. After washing with PBS-T, proteins were incubated with secondary antibody (peroxidase-linked anti-rabbit IgG (NA934, GE Healthcare) or anti-mouse IgG (NA931, GE Healthcare)) diluted in 1% skimmed milk powder for 1 h at RTEnhanced chemiluminescence (ECL) reagent was used to visualise the antibody-bound protein and G:Box F3 (Syngene) was used to image the light signal for the protein bands.
8
Western Blot Analysis of Signaling Proteins
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RIPA buffer (Cell Signaling) was used for cell lysis according to manufacturer’s instructions and the protein concentrations were determined by BCA Protein Assay (Thermo Scientific). Proteins were separated by Criterion SDS-PAGE using Bis-Tris 4–12% gradient polyacrylamide gels (BioRad) and transferred to polyvinylidene difluoride membranes according to standard protocols. The primary antibodies were from Cell Signaling Technology: FOXP3 (Cat. 5298), phospho-SMAD2/3 (Cat. 3101), total SMAD2/3 (Cat. 3102), and β-actin (Cat. 8457). Secondary antibodies were from GE Healthcare: anti-rabbit IgG (NA914) and anti-mouse IgG (NA931). The uncropped scans are provided in Supplementary Figures 3 –5 .
9
Antibody Characterization for Signaling Pathway Analysis
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Anti-IRS-2 (390761) antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-IRS1 (06-248), anti-myosin heavy chain (05-716) and anti-p85 (06-195) antibodies were acquired from Millipore (Billerica, MA, USA). Anti-caspase 3 (#9662), anti-cleaved caspase 3 (#9661), and anti-Bax (#2772) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary anti-rabbit (NA934) and anti-mouse IgG (NA931) antibodies were obtained from GE Healthcare (Pittsburgh, PA, USA). Antibodies were diluted according to the recommendations on their data sheets. Enhanced chemiluminescence (ECL) reagents were acquired from PerkinElmer Life Science (Boston, MA, USA). Alexa Fluor 488 or 594-conjugated secondary anti-mouse, anti-rabbit, or anti-rat IgG antibodies were obtained from Invitrogen (Carlsbad, CA, USA).
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