Equal amounts of concentrated CM (20 µl for 2×105 cells) from BM-MSCs, AT-MSCs and T-MSCs were loaded onto a 5% stacking/10% separating polyacrylamide gel, separated by electrophoresis, transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% skim milk in TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20) and incubated with primary antibodies overnight at 4°C. All primary antibodies were prepared by diluting in 3% BSA (Bovogen Biologicals) and 0.02% sodium azide (Sigma-Aldrich; Merck KGaA) in TBST. Anti-FGF7 mouse monoclonal antibody (sc-365440, 1:200, F-9, IgG1, κ), anti-FLT3L mouse monoclonal antibody (sc-365266, 1:200, F-6, IgG1, κ) and anti-β-actin mouse monoclonal antibody (sc-47778, 1:3,000, C4, IgG1, κ) were purchased from Santa Cruz Biotechnology, Inc. The PVDF membranes were washed 3 times for 10 min in TBST and incubated with goat anti-mouse IgG (H + L)-HRP antibody (#1706516, Bio-Rad Laboratories, Inc.) and diluted in TBST (1:3,000) for 1 h at room temperature. Following incubation, the membranes were washed 3 times for 10 min in TBST and developed using EZ-Western Lumi Femto (DoGenBio Co.). Images were obtained using ImageQuant LAS 500 (GE Healthcare Life Sciences). The pixel densities of the FGF7 and FLT3L bands were divided by the pixel densities of the corresponding β-actin bands for protein quantitation using UN-SCAN-IT-gel 6.1 software.
Ez western lumi femto
Manufactured by DoGenBio
The EZ-Western Lumi Femto is a chemiluminescent western blot detection reagent. It is designed to provide high sensitivity for the detection of target proteins on western blots.
Automatically generated - may contain errors
Lab products found in correlation
Las4000, by GE Healthcare (3 mentions)
Immun blot pvdf membrane, by Bio-Rad (3 mentions)
Bovine serum albumin (bsa), by Bovogen (1 mentions)
Goat anti mouse igg h l hrp antibody, by Bio-Rad (1 mentions)
Mouse anti β actin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Las 4000 apparatus, by GE Healthcare (1 mentions)
Lysis buffer, by iNtRON Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
P smad2 3, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
Ez western lumi pico, by DoGenBio (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
6 protocols using ez western lumi femto
1
Quantitative Analysis of FGF7 and FLT3L Proteins
2
Western Blot Analysis of Protein Samples
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Splenocytes were lysed on ice for 15 min with ice-cold RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS] supplemented with protease inhibitors (1 mM PMSF, 5 μg/ml aprotinin, and 5 μg/ml leupeptin) and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF) and then centrifuged for 5 min at 12,000 rpm at 4°C. The protein concentrations were measured in a BCA protein assay (Pierce, Rockford, IL, USA) using BSA as a standard. Proteins (20 μg/lane) were separated by SDS-PAGE and transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with 5% skim milk for 1 h at room temperature and then washed with Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T). The membranes were incubated overnight at 4°C with specific primary antibodies. After washing with TBS-T, the membranes were incubated for 1 h at room temperature with 5% skim milk containing peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG. Signals were visualized using EZ-Western Lumi Femto (DoGenBio, Seoul, Korea) and quantified on a LAS-4000 (GE Healthcare Life Sciences, Marlborough, MA, USA). The intensity of the protein bands on each blot was quantified by densitometric analysis using ImageJ software (Bethesda, MD, USA) (28 (link), 29 (link)).
3
Western Blot Protein Analysis
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Gastrocnemius and quadriceps muscle samples were homogenized, and then lysed for 20 min in lysis buffer (iNtRON Biotechnology, Seoul, Korea) containing protease and phosphatase inhibitors. Lysate protein concentrations were evaluated in a BCA protein assay (Pierce, Rockford, IL, USA) using BSA as a standard. Equal amounts of proteins were separated by SDS-PAGE gels and transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h in 5% skim milk and then washed in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T). The membranes were incubated overnight at 4°C with indicated primary antibodies. After a washing step, the membranes were blocked for 1 h in 5% skim milk containing peroxidase-conjugated anti-rabbit or anti-mouse. Signals were detected by EZ-Western Lumi Femto (DoGenBio, Seoul, Korea) and visualized using a LAS-4000 apparatus (GE Healthcare Life Sciences, Marlborough, MA, USA). Proteins band intensities on each blot were quantified by densitometric assessment using ImageJ software (Bethesda, MD, USA).
4
Protein Extraction and Western Blot Analysis
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Cells were washed with PBS, and cell lysates were isolated in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitors (1 mM PMSF, 5 μg/mL aprotinin, and 5 μg/mL leupeptin) and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF) and centrifuged at 12,000 rpm for 5 min at 4°C. Protein concentrations were determined by BCA protein assay (Pierce, Rockford, IL, USA) using BSA as the standard. Proteins (20 μg/lane) were separated by SDS-PAGE and transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated at 4°C overnight with specific primary antiserum in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T) and 5% non-fat dry milk. After three washes with TBS-T, membranes were incubated for 1 h with peroxidase-conjugated anti-rabbit or anti-mouse IgG at room temperature. Signals were visualized using EZ-Western Lumi Femto (DoGenBio, Seoul, Korea) and quantified on an LAS-4000 (GE Healthcare Life Sciences, Marlborough, MA, USA). Densitometry of protein bands was performed using the ImageJ software (34 (link)) (Bethesda, MD, USA).
5
Western Blot Analysis Protocol
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Preparation of cell lysates, protein collection and concentration, electrophoresis, SDS-PAGE, and immunoblotting were performed as described preciously [25 (link)]. Primary and secondary antibodies were diluted 1000-fold and 500-fold, respectively. Signals were visualized using an EZ-Western Lumi Femto (DoGenBio, Seoul, Korea) and quantified using a LAS-4000 (GE Healthcare Life Sciences, Marlborough, MA, USA) [26 (link)]. Protein band intensities on each blot were quantified by densitometric analysis using ImageJ 1.48s software (Bethesda, MD, USA).
6
Analyzing Protein Expression in ASCs and Macrophages
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The total protein of ASCs or macrophages was extracted using sample buffer [62.5 mM Tris-HCl, pH 6.8, 34.7 mM sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol, and 10% glycerol], and equal volumes of the protein samples were separated by 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membrane was blocked with 5% skim milk or 5% bovine serum albumin in Tris-HCl-buffered saline containing 0.05% Tween 20 (TBST) for 30 min, and incubated with primary antibodies against TSG-6, Cox-2, IDO2, and GAPDH (Santa Cruz Biotechnology, 1:1000); pp38, SMAD2/3, and cIL-1β (Cell Signaling Technology, 1:1000); and pSmad2/3 (Thermo Fisher Scientific) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibodies (Cell Signaling Technology, 1:5000) for 1 h at room temperature. After primary and secondary antibody incubation, the membranes were washed thrice for 5 min with TBST and then treated with an EZ-Western Lumi Pico or EZ-Western Lumi Femto (DoGenBio, Seoul, Republic of Korea). The target bands were detected using a ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA).
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