Western blot analysis of skin draining LN migDC and cDC. Mice were treated every other day by intraperitonial injection of 10ng recombinant human Flt3L for twelve days prior to LN digestion and FACS sorting. For western blot analysis of protein levels in DCs following in vitro culture, Flt3L derived BMDCs were generated by culture of total bone marrow cells supplemented with 100ng/ml Flt3L for 7 days. Cultures were harvested at 0 hr or treated with 200ng/mL of IFNγ for 24 hr or 48 hr. Western blotting was conducted with the follow antibodies: SOCS2, Abcam; β–actin (AC-15), Sigma. Cells were lysed in modified RIPA buffer containing 50 mM Tris (pH 7.4), 1% NP-40,150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, and protease and phosphatase inhibitor cocktail (Life Technologies). Protein lysates were quantified using protein assay dye reagent (Bio-Rad). Equal amounts of proteins were separated by precast Tris-HCl SDS-polyacrylamide gel (Lonza), transferred to nitrocellulose membrane (Amersham), and incubated with primary antibodies in 1× TBS-T containing 5% non-fat dry milk overnight at 4°C. After washing the membranes in 1× TBS-T, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hr at RT, binding of the primary antibodies were detected using an enhanced chemiluminescence substrate (Life Technologies).
Enhanced chemiluminescence substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan
Enhanced chemiluminescence substrate is a laboratory reagent used to detect and quantify proteins in Western blot analysis. It generates a luminescent signal when it reacts with a specific enzyme, allowing for the visualization and measurement of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (10 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Chemidoc mp imaging system, by Bio-Rad (9 mentions)
Polyvinylidene difluoride membrane, by Merck Group (9 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
Polyvinylidene fluoride membrane, by Merck Group (7 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Image lab software, by Bio-Rad (6 mentions)
Protease inhibitor, by Roche (5 mentions)
Nitrocellulose membrane, by GE Healthcare (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (5 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (4 mentions)
Immobilon p pvdf membrane, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (4 mentions)
174 protocols using enhanced chemiluminescence substrate
1
Western Blot Analysis of Dendritic Cells
2
Protein Expression Analysis of Testes
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Total protein was extracted from the testes with a protein extraction reagent (CWBio, Beijing, China). Western blotting was carried out, as previously described11 . The membranes were exposed to anti‐VDR (1:100), anti‐PPAR‐γ (1:100), anti‐TGF‐β1 (1:500), anti‐NF‐κB (1:500), anti‐phospho‐NF‐κB (1:500), anti‐B‐cell lymphoma 2 (1:500), anti‐Bax (1:1,000), anti‐inhibitor of NF‐κB alpha (1:500) and anti‐β‐actin (1:1,000). After incubation with the horseradish peroxidase‐conjugated secondary antibody, the membranes were treated with enhanced chemiluminescence substrate (Life Technologies, Carlsbad, California, USA) and exposed to X‐ray film. The relative optical density of each target protein was determined and normalized to β‐actin levels using ImageJ software (public domain; National Institutes of Health, Bethesda, Maryland, USA).
3
Quantifying EAAT2 Expression in HFAs
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HFAs were exposed to APV, LPV or DMSO and 12 h after the initial treatment, supernatants were replaced with APV, LPV or DMSO in fresh media. 24 h after exposure, HFAs were lysed in lysis buffer [20 mmol/l Tris, 1% NP-40, 50 mmol/l NaCl, protease inhibitor cocktail set III (1 : 1000, Calbiochem, La Jolla, California, USA)]. Crude protein lysates were separated by 12% SDS-PAGE gel and electro-transferred to nitrocellulose membrane. Membranes were exposed to anti-EAAT2 (Millipore, BilleStrica, Massachusetts, USA) or β-actin (Santa Cruz Biotechnology, Santa Cruz, California, USA) antibodies. Immunoreactive bands were visualized on films using HRP-conjugated secondary antibodies (Jackson Laboratory, Bar Harbor, Maine, USA) and enhanced chemiluminescence substrate (Life Technologies, Carlsbad, California, USA) and quantified using ImageJ (National Institutes of Health). Data from four separate experiments were presented as the mean ± SEM.
4
GTPase Pulldown Assay for Platelet Activation
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The small GTPase pulldown assay was performed using the small GTPase pulldown kit according to the manufacturer's instructions. Briefly, platelets (Arhgef1−/− and WT) were stimulated with thrombin (0.1 U/mL) for 0, 30, 60, 180, and 300 seconds and reactions were stopped with the lysis buffer. The lysates were cleared by centrifugation, and the supernatants were incubated with either rhotekin RBD for RhoA or GST‐humanRalGDSRBD (788–885) for Rap1b for 1 hour at RT. Next, we collected the protein‐bound bead by centrifugation at 3000g for 1 minute. The bead‐bound complexes were washed 3 times with wash buffer. Finally, bead bound‐complexes were eluted using 2× sample buffer, separated by 12% SDS‐PAGE, and transferred to Immobilon‐P PVDF membranes (Millipore Corp). They were then probed with the indicated primary antibodies and visualized with an appropriate horseradish peroxidase–coupled secondary antibody using enhanced chemiluminescence substrate (Thermo Scientific). Images were obtained with a ChemiDoc MP Imaging System (Bio‐Rad). This experiment was repeated 3 times using 3 separate groups, with blood pooled from 8 mice per group. Comparison is based on difference in RhoA and Rap1b activation.
5
Quantitative Western Blot Analysis
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Cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) containing protease inhibitors. Protein concentration was quantified using bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Protein samples (10 µg) were separated on 12% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Roche, Basel, Switzerland). Membranes were incubated in 5% non-fat milk at 25°C for 1 h to block non-specific binding. Membranes were then membranes incubated overnight at 4°C with primary antibodies targeting phosphorylated (p-) Akt (cat. no. ab81283; 1:500; Abcam, Cambridge, MA, USA), total Akt (cat. no. ab18785; 1:500; Abcam), and β-actin (cat. no. ab8226; 1:500; Abcam). Membranes were washed three times with PBS, then samples were incubated with anti-rabbit or anti-mouse secondary antibodies purchased from Beyotime Institute of Biotechnology (cat. no. A0208/A0216; 1:1,000). Bands were visualized using enhanced chemiluminescence substrate (Thermo Fisher Scientific Inc.). Quantity One (version 4.0; Bio-Rad Laboratories, Inc.) was used for densitometric analysis.
6
Chemokine and Galectin Blotting Assay
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100 ng samples of human chemokines or galectins were blotted onto a nitrocellulose membrane, and incubated with 200 nM biotinylated galectins or 120 nM CXCL12 overnight, signals developed with SA‐HRP for 1 h, and added enhanced chemiluminescence substrate (Thermo Fisher Scientific). Densitometric analysis of digital records was performed using the program ImageJ.
7
Western Blot Analysis of Smooth Muscle Markers
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Cell lysate was prepared in modified RIPA buffer (50 mM Tris pH 7.4, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, and 150 mM NaCl) supplemented with protease inhibitors, quantified by Bradford protein assay (Bio-Rad), separated by SDS-PAGE, followed by transferring onto nitrocellulose membranes and blocked with 5% BSA. The membranes were then incubated with primary antibodies including MYH11 (ab53219, Abcam), α-smooth muscle actin (α-SMA) (1A4, Sigma), SM-22 (ab14106, Abcam), KLF4 (PA5–23184, Invitrogen) and GAPDH (14C10, Cell Signaling), followed by incubations with the respective horseradish peroxidase-conjugated secondary antibodies. Blots were developed using enhanced chemiluminescence substrate (ThermoFisher) and imaged in a gel documentation system (Alpha Innotech), followed by quantification using ImageJ.
8
Western Blot Protein Expression Analysis
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Whole cell extracts were prepared by lysing cells in lamelli 2× sample buffer containing a cocktail of phosphatase inhibitor, phosSTOP (Rhoche) and protease inhibitors. The total lysate of proteins was separated on 6 to 12% SDS-PAGE by electrophoresis. Proteins were transferred to nitrocellulose membrane, and blocked with 5% powdered skim milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.01% Tween). The membrane was then incubated with indicated primary antibodies diluted into 5% BSA in TBST. After primary antibody incubations, membranes were incubated with HRP-conjugated anti-mouse or rabbit IgG secondary antibodies diluted into 5% skim milk in TBST for 1 h. Between each steps, membranes were washed with TBST extensively more than 3 times at 5 min. Proteins were visualized with enhanced chemiluminescence substrate (Thermoscientific). Chemi-doc MP (Bio-rad, Inc., USA) was used to capture images. α-tubulin expression was evaluated as a protein loading control. Protein expression was analyzed by densitometry analysis program, ImageJ Software (National Instituties of Health, USA).
9
Protein Expression Analysis in GC-1 Cells
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GC-1 cells were lysed in RIPA buffer (Millipore, USA) with 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml CompleteTM EDTA free protease inhibitor cocktail (Roche, USA) and phosphatase inhibitors (5 mM sodium orthovanadate). Protein lysates were loaded on to SDS-PAGE gels, transferred to nitrocellulose membranes (Amersham Biosciences, Germany), immunoblotted with antibodies and visualized using enhanced chemiluminescence substrate (Thermo Fisher Scientific, USA). Protein levels of p53, p21 and γ-H2AX were normalized to GAPDH. The primary antibodies in this study were the following: anti-GAPDH (Cell Signaling Technology, USA), anti-p53 (Santa Cruz, USA), anti-γ-H2AX (Abcam, USA) and anti-p21 (Santa Cruz).
10
Western Blot Analysis of Liver Proteins
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Liver samples were homogenized in ice‐cold RIPA buffer (50 mM Tris‐HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholic acid, and 1% NP‐40) in the presence of protease and phosphatase inhibitor cocktails (Thermo‐Fisher). Total sample lysates were centrifuged for 15 minutes at 10 163 g and supernatant were mixed with 6x reducing SDS buffer (Alfa‐Aesar) and boiled at 100°C for 5 minutes. Protein samples (30 μg) were loaded and separated on gradient SDS‐PAGE gels (Bio‐Rad) and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% non‐fat milk in 1× TBS supplemented with 0.1% Tween20 (Bio‐Rad) and incubated with the following antibodies: GLS2 (Novus Biologicals, NBP1‐76544), GLUD1 (ThermoFisher Scientific, PA5‐19267), OTC (Abcam, ab203859), and GAPDH (Cell Signaling, 2118 L). Bound antibodies were detected using horseradish peroxidase‐conjugated anti‐rabbit or anti‐goat secondary antibodies (1:10 000; Jackson ImmunoResearch) and enhanced chemiluminescence substrate (Thermo‐Fisher). Band intensities were quantified with ImageJ software.
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