Cell lysates containing equal amounts of protein were electrophoresed on 12% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted using primary antibodies. Anti-vimentin (VI-01), anti-β-catenin (ab32572), anti-Twist (ab5087), anti-E-cadherin (ab1416), and anti-cytokeratin-18 (C-04) antibodies were purchased from Abcam (Cambridge, UK). Anti-N-cadherin (13A9) and anti-tubulin (DM1A) antibodies were obtained from Calbiochem (Darmstadt, Germany). Anti-Snail (3879) was obtained from Cell Signalling Technology (Danvers, MA). Following horseradish peroxidase-conjugated secondary antibody incubation, immunoblots were detected using the Immun-Star™ HRP Chemiluminescent kit (Bio-Rad, Hercules, CA).
Immun star hrp chemiluminescent kit
Manufactured by Bio-Rad
Sourced in United States
The Immun-Star HRP Chemiluminescent Kit is a laboratory equipment product designed for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent substrate to produce a light signal proportional to the amount of target protein present in the sample. The kit provides the necessary reagents and components to perform this analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Hybond p membrane, by GE Healthcare (2 mentions)
Hybond, by Cytiva (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Aar 013, by Alomone (2 mentions)
Ab108252, by Abcam (2 mentions)
Ab97057, by Abcam (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Versadoc 4000 mp imager, by Bio-Rad (1 mentions)
Hrp linked anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Anti mouse igg hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Bead beater, by Biospec (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Donkey anti goat igg hrp sc 2020, by Santa Cruz Biotechnology (1 mentions)
Chicken anti mouse igg hrp sc 2954, by Santa Cruz Biotechnology (1 mentions)
Ab133303, by Abcam (1 mentions)
14 protocols using immun star hrp chemiluminescent kit
1
Immunoblotting Analysis of EMT Markers
2
Western Blot Protein Analysis
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Western blot analyses were carried out using crude cell lysates of cell lines or testicular cancer samples heated for 5 min at 95°C in 2x SDS sample buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% Bromophenol Blue, 20% glycerol, 200 mM DTT). Proteins were separated in 10% polyacrylamide gel and transferred to Hybond-P membrane (GE Healthcare, UK).
Treatment with primary antibodies listed inTable S1 was followed by the addition of either secondary anti-rabbit IgG HRP-linked antibody (Cell Signaling, USA, #7074S) or secondary anti-mouse IgG HRP-linked antibody (Cell Signaling, USA, #7076S). The membrane was visualized using the Immun-Star HRP Chemiluminescent kit (Bio-Rad, USA) and bands were detected either in VersaDoc MP4000 imager (Bio-Rad, USA) or on X-ray film. Technical duplicates were used to ensure reproducibility.
Treatment with primary antibodies listed in
3
Immunoblotting of M. smegmatis Ald Protein
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Bacterial cells were collected and lysed using Bead Beater (BioSpec Products) and heated for 5 min at 95°C in 2× sodium dodecyl sulfate (SDS) sample buffer (100 mM of Tris–HCl, pH 6.8, 4% SDS, 0.2% Bromophenol Blue, 20% glycerol, and 200 mM of DTT). Protein was measured by the Bradford assay. Equal amounts of total protein (5 μg each) were resolved by SDS-PAGE in a 12% gel and transferred onto Hybond-P membranes (GE Healthcare, UK), which were blocked with 5% w/v nonfat dry milk (Bio-Rad, USA), and incubated with primary antibodies against l-alanine dehydrogenase (Ald) (LS-C184923/70850, 1:10,000; LSBio, USA) and then with secondary anti-rabbit horseradish peroxidase (HRP)-conjugated IgG (Cell Signaling Technology, USA). Specific signals were visualized on X-ray films (Retina, USA) using the Immun-Star HRP Chemiluminescent kit (Bio-Rad) and quantified by densitometry using the GelPro software. The specificity of the LS-C184923/70850 antibody to the Ald protein of M. smegmatis was additionally confirmed by reaction with recombinant Ald (Suppl. Materials and Methods Suppl. Figure 1
4
GR Protein Expression Analysis
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MRC-5 cells were lysed with RIPA buffer (Sigma) containing proteinase inhibitor cocktail and quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). Proteins were separated on a 13.5% SDS-PAGE gel, blotted and probed with GR antibody (611226, BD Biosciences). The membranes were visualised post incubation with Bio-Rad Immun-Star™ HRP Chemiluminescent Kit. Total protein content was determined by staining the GR protein nitrocellulose membrane with 1x Ponceau S Staining solution for 5 min followed by a 30 s wash in destaining solution. Densitometric analyses of the GR protein band intensity was calculated relative to GAPDH protein.
5
Mitochondrial Protein Quantification and Analysis
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Protein concentration in the isolated mitochondria from the nigral region (n=4) and whole homogenate (n=4) were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Fremont, CA, USA). Equal amounts of protein were separated on a 5–10% bis-tris polyacrylamide gel, and transferred to nitrocellulose membranes. Membranes were incubated overnight with primary antibodies against VDAC/porin (V2139, 1 : 1000) and α-Tubulin (T5168, 1 : 50 000) from Sigma, HDAC2 (sc-56685, 1 : 200), AT1 (sc-31181, 1 : 200) and AT2 (sc-9040, 1 : 200) from Santa Cruz Biotechnology and rabbit monoclonal antibody to Nox4 (ab133303, 1 : 800) from Abcam. The following HRP-conjugated secondary antibodies were used: Protein A (1 : 5000) (NA9120V; GE Healthcare), chicken anti-mouse IgG-HRP (sc-2954, 1 : 2500) and donkey anti-goat IgG-HRP (sc-2020, 1 : 2500) from Santa Cruz Biotechnology. Immunoreactive bands were detected with an Immun-Star HRP Chemiluminescent Kit (170-5044; Bio Rad) and visualized with a chemiluminescence detection system (Molecular Imager ChemiDoc XRS System; Bio Rad). Specificity of the antibodies was confirmed as indicated above.
6
Western Blot Analysis of ACE2 and RAS Receptors
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Homogenates from rat lung tissue or A549 cells were lysed in RIPA buffer containing PMSF (Sigma) and protease inhibitor cocktail (Sigma). Total proteins were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). An equal amount of protein lysates or cell culture medium was separated on 8–10% Bis-Tris polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were incubated overnight at 4°C with primary antibodies against the extracellular domain of ACE2 (ab108252; Abcam; 1:1000), AT1 (sc-31181; Santa Cruz; 1:200), AT2 (sc-9040; Santa Cruz; 1:200) and Mas (AAR-013; Alomone labs; 1:1000) receptors. Membranes were reincubated with loading controls: anti-α-tubulin (T5168; 1:50.000; Sigma) or GAPDH (G9545; 1:25.000; Sigma), or ponceau (Sigma, P7170-1L). The following horseradish peroxidase (HRP)-conjugated secondary antibodies were used: goat anti-rabbit-HRP and goat anti-mouse-HRP (Santa Cruz Biotechnology; 1:2500). Bound antibody was detected with an Immun-Star HRP Chemiluminescent Kit (Bio-Rad; 170-5044) and visualized with a chemiluminescence detection system (Bio-Rad; Molecular Imager ChemiDoc XRS System). The data were then expressed relative to the value obtained for the control to counteract possible variability among batches.
7
Quantifying AT1-AA Autoantibodies by ELISA
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Serum and CSF AT1-AA levels were measured using a specific solid-phase, sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of these autoantibodies (Catalog Number 12,000; Cell Trend; Luckenwalde, Germany). The manufacturer’s instructions were followed, with a light modification as previously described [52 (link)] consisting of the use of an HRP-conjugated goat anti-rat (1:2500, ab97057, Abcam, Cambridge, UK) as a secondary antibody. Immunoreactivity was detected with an Immun-Star HRP Chemiluminescent Kit (170–5044, Bio-Rad) and luminescence was measured using an Infinite M200 multiwell plate reader (TECAN, Männedorf, Switzerland).
8
Western Blot Analysis of TLR3 Expression
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Cells were homogenized in RIPA lysis buffer containing a 1% protease inhibitor cocktail (Sigma-Aldrich), 5 μM EDTA, and 200 μM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride to extract the total protein. Equal amounts of protein homogenates in the samples were separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA), transferred onto polyvinylidene difluoride membranes (pore size 0.45 μM, Bio-Rad), blotted with human monoclonal antibody against TLR3 (1:1000, Cell Signaling Technology, Beverly, MA, USA) or GAPDH (1:1000, Cell Signaling Technology), and probed with the corresponding peroxidase-conjugated secondary antibodies (KPL, Gaithersburg, MD, USA). Signals from bound antibodies were detected using an Immun-Star HRP Chemiluminescent kit (Bio-Rad).
9
Western Blot Analysis of Immune Markers
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Spleen tissue was dissected and sonicated in T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Inc.) containing protease cocktail (Beyotime Institute of Biotechnology) on ice. The lysates were centrifuged at 10,000 × g for 10 min at 4°C. Protein concentration was determined using a BCA assay kit (Beyotime Institute of Biotechnology). Equal amounts of extracted protein samples (50 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk at 37°C for 1 h and incubated overnight at 4°C with the following primary antibodies: Anti-GATA-3 (1:1,000), anti-T-bet (1:1,000), anti-STAT-3 (1:1,000), anti-Foxp3 (1:2,000) and anti-β-actin (1:5,000). Subsequently, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (cat no. A0208) or goat anti-mouse IgG (cat no. A0258) secondary antibodies (1:2,000; Beyotime Institute of Biotechnology) at 37°C for 2 h. Protein bands were visualized using the Immun-Star™ HRP Chemiluminescent kit (Bio-Rad Laboratories, Inc.) and the Molecular Imager® VersaDoc™ MP imaging system (Bio-Rad Laboratories, Inc.). β-actin served as the loading control.
10
Protein Quantification and Western Blotting
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The protein content of cell extracts was determined by a Bradford assay (Bio-Rad, Hercules, CA, USA). A total of 20–30 μg protein was electrophoresed by 10–15% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated with the primary antibodies at the appropriate concentrations, and subsequently incubated with HRP-conjugated goat anti-rabbit or goat anti-mouse IgGs (1:2,000; Beyotime Institute of Biotechnology). Labeled bands were detected by the Immun-Star™ HRP Chemiluminescent kit (Bio-Rad), and images were captured and the intensity of the bands was quantified by the VersaDoc™ image system (Bio-Rad, Regents Park, Australia).
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