Western blotting analysis was performed as described previously.17 Proteins were fractionated using NuPAGE 4% to 12% Bis‐Tris gels (Invitrogen) and transferred to Protran nitrocellulose transfer membrane (Whatman, Japan). The membrane was blocked using PBS containing 5% nonfat milk for 30 minutes at room temperature and incubated with the primary antibody (anti‐ABCA1: 1:2000, NB400‐105; anti‐ABCG1 (anti‐ATP binding cassette G1): 1:2000, NB400‐132 [Novus Biologicals, CO], anti–carnitine palmitoyltransferase Ia: 1:2000, ab128568 [Abcam, UK]; anti–β‐actin: 1:3000, A5441 [Sigma‐Aldrich]) overnight at 4°C. The membrane was washed in PBS containing 0.05% Tween‐20, and then it was incubated with the secondary antibody (anti‐rabbit IgG horseradish peroxidase linked: 1:2000; anti‐mouse IgG horseradish peroxidase linked: 1:2000 [GE Healthcare, UK]) for 30 minutes at room temperature. The membrane was washed in PBS containing 0.05% Tween‐20 and incubated with ECL Western Blotting Detection Reagent (GE Healthcare) for 5 minutes; then, the signal was detected using an LAS‐4000 system (Fuji Film, Japan).
Horseradish peroxidase linked anti mouse igg
Manufactured by GE Healthcare
Sourced in United States
Horseradish peroxidase-linked anti-mouse IgG is a secondary antibody conjugate used in various immunoassay techniques. It contains horseradish peroxidase enzyme attached to an antibody that specifically recognizes mouse immunoglobulin G (IgG). This conjugate can be used to detect and quantify the presence of mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting detection reagent, by GE Healthcare (4 mentions)
96 well microtiter plate, by Corning (3 mentions)
Mouse monoclonal anti pcna, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase linked anti rabbit igg, by GE Healthcare (2 mentions)
Horseradish peroxidase linked anti mouse iga, by Southern Biotech (2 mentions)
Ab128568, by Abcam (1 mentions)
Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Protran nitrocellulose transfer membrane, by Cytiva (1 mentions)
Nb400 132, by Novus Biologicals (1 mentions)
Anti rabbit igg horseradish peroxidase, by GE Healthcare (1 mentions)
A5441, by Merck Group (1 mentions)
Nb400 105, by Novus Biologicals (1 mentions)
Las 4000 system, by Fujifilm (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Anti his tag antibody, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Anti his tag antibody, by GenScript (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
11 protocols using horseradish peroxidase linked anti mouse igg
1
Western Blotting Protein Quantification
2
Western Blot Analysis of Protein Expression
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Proteins were separated by SDS-PAGE and transferred to a PVDF membrane by dry transfer using the iBlot dry blotting system from Invitrogen (Waltham, MA, USA). Membranes were blocked for 1 h at room temperature in 5% w/v milk or BSA in TBS-T (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 0.05% Tween), followed by incubation with primary antibody overnight at 4 °C. Primary antibodies: anti-His-tag antibody (#A00186, GenScript, 1:5000) (Piscataway, NJ, USA), anti-His-tag antibody (#12698, Cell Signaling, 1:1000) (Danvers, MA, USA), anti-NEIL2 antibody (#124106, Abcam, 1:500) (Cambridge, UK), anti-P35 antibody (anti-p35, SantaCruz, #sc-518009, 1:2500) (Dallas, TX, USA), and anti-actin antibody (#A2228, Sigma, 1:10,000) (Merck, Darmstadt, Germany). Subsequently, the membrane was washed in TBS-T and incubated with secondary anti-mouse IgG Horseradish Peroxidase Linked (GE Healthcare, #NA931, 1:5000) (Chicago, IL, USA) or anti-rabbit IgG Horseradish Peroxidase Linked (GE Healthcare, #NA934, 1:5000) (Chicago, IL, USA) antibody for 1 h at room temperature, followed by additional washing in TBS-T and detection with ECL Prime Western Blotting Detection Reagent from GE Healthcare (Chicago, IL, USA) according to the manufacturer’s protocol.
3
PCNA Protein Expression in ht-UtLM Spheroids
4
PCNA Protein Expression in ht-UtLM Spheroids
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Protein extractions and western blotting were performed to determine the protein expression of PCNA in ht‐UtLM spheroids with or without TBBPA treatment at 10−3 μM for 7 days. The primary antibody mouse monoclonal anti‐PCNA (#2586, Cell Signaling) was used at 1:2000 dilution over night at 4°C. Secondary antibody anti‐mouse IgG horseradish peroxidase linked (A931V, GE Healthcare) was used at 1:5000. The detection kit used was the ECL Western Blotting Detection Reagent (#RPn2106, GE Healthcare). Band intensity was quantified by Fiji.
5
Quantification of anti-flagellin antibodies
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Quantification of anti-flagellin- specific IgA and IgG has been previously described (34 (link)–36 (link, link)). Briefly, 96-well microtiter plates (Costar, Corning, NY) were coated with 100 ng/well of laboratory-made flagellin in 9.6 pH bicarbonate buffer overnight at 4 °C. Serum samples from mice were then applied either pure or at a 1:100 dilution for 1 h at 37 °C. After incubation and washing, the wells were incubated with either horseradish peroxidase-linked anti-mouse IgG (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania) or horseradish peroxidase-linked anti-IgA (Southern Biotech, Birmingham, Alabama). Quantification of immunoglobulin was then developed by the addition of 3,3′,5,5′-Tetramethylbenzidine and the optical density was calculated by the difference between readings at 450 nm and 540 nm.
6
IgA and IgG Quantification of Anti-Flagellin
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Quantification of anti-flagellin- specific IgA and IgG has been previously described32 (link)–34 (link). In brief, 96-well microtiter plates (Costar, Corning, New York) were coated with 100 ng/well of either laboratory-made Salmonella Typhimurium-derived or purchased B. subtilis-derived flagellin in 9.6 pH bicarbonate buffer overnight at 4 °C. Serum or fecal samples from mice were then applied either pure or at a 1:100 dilution for 1 h at 37 °C. After incubation and washing, the wells were incubated with either horseradish peroxidase-linked anti-mouse IgG (GE Healthcare Life Sciences, NA931V) or horseradish peroxidase-linked anti-mouse IgA (Southern Biotech, 1040–05). Quantification of immunoglobulin was then developed by the addition of 3,3′,5,5′-Tetramethylbenzidine and the optical density was calculated by the difference between readings at 450 nm and 540 nm.
7
Western Blotting for CD200 Expression
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Cells were lysed in whole-cell extraction buffer (20 mM HEPES-NaOH, 0.5% NP-40, 15% glycerol) containing a complete protease inhibitor cocktail (Roche, Basel, Switzerland). The proteins were separated in a 12% SDS-polyacrylamide gel and transferred to an Immobilon-P PVDF membrane (Merck Millipore). Blots were incubated for 1 h at RT with a mouse monoclonal antibody recognizing human CD200 (Proteintech, Rosemont, IL, USA) or a mouse polyclonal antibody recognizing human β actin (Santa Cruz). After washing in TBS-T, the membranes were incubated for 1 h at RT with horseradish peroxidase-linked anti-mouse IgG (GE healthcare, Chicago, IL, USA). ECL Western Blotting Detection Reagents (GE Healthcare) were used to develop the high-performance chemiluminescence film (GE Healthcare).
8
Western Blot Analysis of Signaling Proteins
9
Lipoylated Protein Analysis in Parasites
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EPI pellets were washed twice with PBS and resuspended in lysis buffer (0.5% Nonidet P-40, 500 mM NaCl, 5 mM EDTA, 1 mM DTT, 50 mM Tris-Base, 0.4% SDS, pH 7.4) at 1 × 106 parasites/μl. Samples were sonicated using 3 pulses of 30 s at 100% amplitude (Q700 sonicator, QSONICA), with 15 s breaks between to rest the tubes on the ice. Samples were centrifuged at 16,000g for 20 min and the supernatant was collected. Equivalent to 1 × 106 parasites were resolved by electrophoresis on a Mini-Protean TGX precast gel (Bio-Rad). Lipoylated proteins were detected with rabbit antilipoic acid antibody (ab 58,724, Abcam). Bound antibodies were detected with horseradish peroxidase-linked anti-rabbit IgG (GE Healthcare) and ECL Prime Western Blotting Detection Kit (GE Healthcare). The loading control was performed with mouse anti-α-tubulin (Sigma-Aldrich) and bounded antibodies were detected with horseradish peroxidase-linked anti-mouse IgG (GE Healthcare). Quantification was performed after normalization of lipoic acid signal to tubulin, using FIJI (57 (link)). Each experiment was carried out in at least three independent experiments.
10
Protein Expression and Tissue Microarray Analysis
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Cytoplasmic protein extracts were prepared and western blotting performed as described [21 (link)]. Briefly, protein samples were resolved in NuPAGE 4-12% Bis Tris gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Roche). Membranes were blocked in Tris-buffered saline/Tween 20 (TBST)/5% skim milk and probed with β-actin [AC-15] (Abcam ab6276; 1:10000) and RALA (Cell Signaling Technologies #4799; 1:1500). Detection was performed using horseradish peroxidase-linked anti-mouse IgG (GE Healthcare; Cat #NA931V) or anti-rabbit IgG (GE Healthcare; Cat #NA934V) with Luminata Classico Western HRP substrate (Millipore #WBLUC0100) and ECL-Hyperfilm (VWR #GE HE28-9068-37). Tissue microarray (TMA) slides (Cat. #PR243a) obtained from US Biomax, Inc. (Rockville, USA; Supplementary Table 1 ). Sections were de-paraffinized in xylene, rehydrated through graded alcohols and subjected to antigen retrieval in citrate buffer pH 6.0 under pressure. Sections were incubated with a RALA antibody (Cell Signaling Technologies #4799; 1:100) for 60 minutes and immunoreactivity visualized using a Dako Envision+ Dual link system-HRP (30 min) and diaminobenzidine (DAKO). Stained slides were independently scored by three researchers.
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