Total proteins were extracted from lung tissues or cells. Lung or cultured fibroblasts were lysed with radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.5) and centrifuged at 14,000 × g for 10 min at 4°C. Protein concentrations in supernatants were measured with a Bradford assay (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Protein samples (20 µg/lane) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4°C with the primary antibody against Col I, α-SMA, Fn, ACE and AT1 (all 1:500) followed by a peroxidase-labeled affinity-purified secondary antibody to rabbit/mouse IgG (1:5,000, cat. no. 074-1506, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA) for 2 h at 37°C. Target bands were visualized by the addition of an Enhanced Chemiluminescence Prime Western Blotting Detection reagent (GE Healthcare, Chicago, IL, USA). The density of specific bands was analyzed using Image-Pro Plus image processing software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) and the results were normalized to β-actin or GAPDH (both 1:500).
Enhanced chemiluminescence prime western blotting detection reagent
Manufactured by GE Healthcare
Sourced in United States
Enhanced chemiluminescence prime western blotting detection reagent is a laboratory equipment product that provides a sensitive and reliable method for detecting and quantifying proteins in western blot analyses. It utilizes chemiluminescence technology to enable the detection of target proteins with high sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Bradford assay, by Solarbio (1 mentions)
Imagequant tl software package, by GE Healthcare (1 mentions)
Imagequant las 4000 mini imager, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Nitrocellulose transfer membrane, by Bio-Rad (1 mentions)
Nonfat milk powder, by Cell Signaling Technology (1 mentions)
Ponceau s solution, by Merck Group (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by GE Healthcare (1 mentions)
Ibright 1500, by Thermo Fisher Scientific (1 mentions)
Hmgn3, by Abcam (1 mentions)
Tsbp1, by Abcam (1 mentions)
Hmgn2, by Cell Signaling Technology (1 mentions)
Foxa1, by Cell Signaling Technology (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using enhanced chemiluminescence prime western blotting detection reagent
1
Quantifying Lung Fibrosis Proteins
2
Protein Analysis by Western Blotting
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The cell and virus samples were dissociated in Laemmli buffer (2% SDS, 100 mM DTT, 125 mM Tris-HCl, pH 6.8), heated at 90°C for 5 min, and electrophoresed on 12% SDS-polyacrylamide gels. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) (0.2-μm pore size). Membranes were incubated overnight at 4°C with the primary antibodies and for 1 h at RT with the corresponding secondary antibodies conjugated to horseradish peroxidase (GE Healthcare). Blots were developed with enhanced chemiluminescence Prime Western blotting detection reagent (GE Healthcare), imaged with an ImageQuant LAS 4000 mini imager (GE Healthcare), and quantified with the ImageQuant TL software package (GE Healthcare).
3
Monitoring αS Aggregation by Immunoblotting
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The monomeric αS solution (100 μM) in a 2-ml protein-low binding tube (Sarstedt, Princeton, NJ) was incubated at 37 °C and 30 rpm. An aliquot of the solution was sampled at each time point and was blotted on a nitrocellulose transfer membrane (0.2 μm pore size; Bio-Rad). The membranes were probed with the anti-αS monoclonal (mAb#10-8), the anti-oligomer A11 polyclonal (Thermo Fisher Scientific, MA, USA), or the anti-fibril monoclonal (mAb#B7-5) antibodies51 (link), followed by incubation with the horseradish peroxidase-conjugated secondary antibodies. Immunoreactive signals were developed using an enhanced chemiluminescence prime western blotting detection reagent (GE Healthcare, Milwaukee, WI). The signals were visualized using a LuminoGraph I imaging system (ATTO Corporation, Tokyo, Japan).
4
Western Blot Analysis of Neural Markers
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After quantification of the protein concentration using the Pierce BCA assay (Thermo Scientific, USA), protein lysates were separated on 12.5% SDS polyacrylamide gels and transferred to nitrocellulose membranes (0.2 µm, Amersham, USA) at 4 °C overnight. Equal loading of 7.5-µg protein per lane and transfer of proteins were confirmed by staining of membranes with Ponceau S solution (Sigma Aldrich). Nonspecific binding was blocked by incubation in 5% nonfat milk powder (Cell Signaling, USA) and 0.1% Tween-20 in tris-buffered saline (TBS) followed by incubation with the primary antibodies. The following primary antibodies were applied: rabbit anti-NeuN, goat anti-vascular cell adhesion molecule-1 (VCAM-1), rabbit anti-Iba-1, mouse anti-GFAP, and rabbit anti-glutaraldehyde-3-phosphate dehydrogenase (GAPDH) (Suppl. Table 7 ), each in blocking solution at 4 °C overnight. Membranes were incubated with appropriate peroxidase-conjugated secondary antibodies (all 1:5000, Dako, Denmark) in blocking solution at room temperature for 1 h followed by chemiluminescent detection with the enhanced chemiluminescence prime Western blotting detection reagent (Amersham, GE Healthcare Life Science, USA). For visualization and densitometric analysis, the ChemiDocXRS + imaging system and ImageLab software (Bio-Rad, Germany) were used.
5
Western Blot Analysis of Phospho-S6 Ribosomal Protein
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All samples were diluted to 1 mg/mL using 4× Laemmli Sample Buffer for SDS‐PAGE with Tris‐glycine gels (12%). Samples were transferred to polyvinylidene fluoride membranes (GE Healthcare), and blocked for 1 hour with 5% non‐fat milk diluted in 1XPBS on a rocking platform at RT. Incubation with primary antibodies was done overnight at 4°C: p‐S6 (Ser240/244) (rabbit, 1:1K; #2215; Cell Signaling), p‐S6 (Ser235/236) (rabbit, 1:1K; #4858; Cell Signaling), S6 (mouse, 1:1K; #2317; Cell Signaling), and Actin (rabbit, 1:1K; A2066; Millipore Sigma). Membranes were washed in 1XPBS‐0.1%Tween and incubated with HRP‐linked secondary antibodies (anti‐rabbit or anti‐mouse, 1:1K; #7074, #7076; Cell Signaling). Immunoreactive bands were visualized using enhanced chemiluminescence prime western blotting detection reagent (GE Healthcare), captured on Double Emulsion Blue Autoradiography Film (Midwest Scientific). Membranes were stripped from primary antibodies using stripping buffer (25 mM glycine, pH 2.0, 10% SDS) and re‐blotted as described above. Note that there was not sufficient protein from P5 for immunoblotting. Image J software (NIH) was used to measure the relative signal intensity of immunoreactive bands for phosphorylated and total proteins for each sample/lane.37 Actin was used as a loading control.
6
Western Blot Analysis of Transcriptional Regulators
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Tissue sample (100 µg) was added to the RIPA buffer (Thermo, Waltham, MA, USA) for homogenization and then subjected to centrifugation to obtain the supernatant. Protein concentration was quantified using the BCA assay kit (Thermo, Waltham, Massachusetts). The tissue lysates were separated using 10% SDS–PAGE gel for 90 min and transferred to polyvinylidenefluoride membranes (Roche, Basel, Switzerland) for 2 h. After blocking with 5% BSA in Tris-buffered saline for 1 h. Then, the blots were incubated with primary antibodies for FoxA1 (Cell signaling, Beverly, MA, USA), HMGN1 (Cell signaling, Beverly, MA, USA), HMGN2 (Cell signaling, Beverly, MA, USA), HMGN3 (Abcam, Cambridge, UK), and TSBP1 (Abcam, Cambridge, UK) overnight, after which they were reacted with an HRP-conjugated secondary antibody for 2 h. An enhanced chemiluminescence prime western blotting detection reagent (GE Healthcare, Chalfont St. Giles, UK) was used to visualize the protein bands. The density of respective bands was analyzed with the iBright 1500 (Thermo, Waltham, MA, USA).
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