Activation of the STAT3 pathway was assessed by quantifying pSTAT3tyr705 using immunoblot analysis with detection of fluorescent signals using an infrared fluorescence scanner (Licor Biosciences, Lincoln, NE) or with detection using an ECL chemiluminescent substrate (Amersham Biosciences, Piscataway, NJ) captured onto x-ray film (Fuji Medical systems, Stamford, CT) as described previously [12] (link), [19] (link). Briefly, following incubation with primary antibodies (anti-phospho-STAT3tyr705[1∶500]), blots were washed with phosphate buffered saline with 0.1% Tween 20 and incubated with fluorescent-labeled secondary antibodies (1∶2500) for 1 h prior to scanning by Licor or using a Personal Densitometer (Molecular Dynamics, Sunnyvale, CA).
Personal densitometer
Manufactured by GE Healthcare
Sourced in United States
The Personal Densitometer is a compact, user-friendly device designed to measure bone mineral density (BMD). It utilizes dual-energy X-ray absorptiometry (DXA) technology to provide accurate and reliable assessments of bone health. The device is intended for use in clinical settings to support the diagnosis and management of conditions related to bone density, such as osteoporosis.
Automatically generated - may contain errors
Lab products found in correlation
Infrared fluorescence scanner, by LI COR (1 mentions)
Ecl chemiluminescent substrate, by Cytiva (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Xar 5 film, by Kodak (1 mentions)
Bcl2l1, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Carl Roth (1 mentions)
Fusion fx imaging system, by Avantor (1 mentions)
Erk1 2 and p38, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by GE Healthcare (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Anti β actin, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Hyperfilm ecl, by GE Healthcare (1 mentions)
Peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
8 protocols using personal densitometer
1
Quantification of Activated STAT3 Pathway
2
Western Blot Protein Detection
3
Western Blot Analysis of Protein Expression
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As described in a previous paper [12 (link)], the T lymphocytes with different conditions were harvested and lysed on ice with protein lysate containing Phenylmethylsulphonylfluoride (PMSF) for 30 min. The protein concentration was determined by using the Bradford protein assay with BSA as standard. Total proteins (20 μg) were subjected to 10% SDS-PAGE and blotted onto polyvinylidene fluoride membrane at 4 V for 20 min. After being blocked in TBS-T containing 5% (w/v) skimmed milk at 37 °C for 1 h, the membranes were then incubated overnight at 4 °C with various antibodies: anti-CaSR (1:800), anti-Caspase-12 (1:800), and anti-P-p65 (1:1000), respectively, then incubated with anti-IgG antibody conjugated with horseradish peroxidase diluted 1:2000 in TBS-T for 1 h at room temperature. Antibody–antigen complexes were detected by using Western Blue® ECL Plus kit (Beyo, Shanghai, China). Anti-β-actin IgG (Beijing, China) was used (1:400) as the house-keeping internal control. Quantitative comparisons of various proteins under various experimental conditions were performed using a Personal Densitometer™ (Molecular Dynamics, Bio-Rad, Hercules, CA, USA).
4
RGNNV Infection and Apoptosis Regulation in GF-1 Cells
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GF-1 cells were cultured in 60 mm Petri dishes (10 5 /mL) for 20 h to monolayer confluence, rinsed twice with PBS, treated with 3-methyladenine (3-MA, 2 mM) for 2 h, and then infected with RGNNV (MOI = 1) for 0, 24, 36 or 48 h at 28 • C. In other experiments, Flag, Flag-Bcl2, and Flag-BCL2L1-producing GF-1 cells were cultured in 60 mm Petri dishes (10 5 /mL) for 20 h to monolayer confluence, rinsed twice with PBS, and then infected with RGNNV (MOI = 1) for 0, 24, 36 or 48 h at 28 • C. At the end of each incubation period, the culture medium was aspirated, and the cells were washed with PBS and lysed in 0.3 mL of lysis buffer (10 mM Tris, pH 7.3, 20% glycerol, 10 mM SDS, 2% ß-mercaptoethanol, pH 6.8). Lysates were separated by SDS-PAGE [29] (link) and the proteins were transferred to nitrocellulose. Blots were incubated with polyclonal antibodies to protein α, protein B2, mouse BCL2 (Cell Signaling, 15071, Danvers, MA, USA), BCL2L1 (Cell Signaling, 2764, Danvers, MA, USA), LC3 (GeneTex, GTX127375, Irvine, CA, USA), and ACTB/ßactin, followed by peroxidase-labeled goat anti-rabbit conjugate (1:7500) (Calbiochem, MAB1501, Darmstadt, Germany). Binding was detected by chemiluminescence; the signals were recorded on Kodak XAR-5 film (Eastman Kodak, Rochester, NY, USA) [30] . Protein expression was quantified using the Personal Densitometer (Molecular Dynamics).
5
Protein Separation and Detection Protocol
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For SDS-PAGE 12.5% PAA gels were used. Proteins were blotted to PVDF membrane (Carl-Roth). The membranes were blocked with 5% skimmed milk in TBST and incubated over night with the primary antibody diluted 1:500-1:5,000 in 5% skimmed milk. The peroxidase conjugated secondary antibodies were incubated for 1 h diluted 1:10,000 in 5% skimmed milk. Blots were developed using the ECL kit and films from GE Healthcare. Bands were scanned using a personal densitometer (GE Healthcare).
6
Retinal Protein Expression Analysis
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24 h after IRI retinal tissue was harvested for analysis of protein expression. Total protein from ¾ of retina was extracted and processed for Western Blot as described previously. The contralateral eyes were processed in the same manner. The membranes were blocked with 5% skim milk in Tween20/PBS and incubated in the recommended dilution of protein specific antibody (p-ERK1/2 #4370, p-p38 #9211, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Freiburg, Germany), proteins were visualized using the ECL Western blotting detection reagent (Western Lightning plus ECL, #NEL103001EA, PerkinElmer, Waltham, MA, USA) following the manufacturer´s instructions. Images were generated with Fusion Fx imaging system (PEQLAB Biotechnologie GmbH, Erlangen, Germany). For normalization, blots were re-probed with ERK1/2 and p38 (#4695, #9212, Cell Signaling Technology, Danvers, MA, USA). Blots were analyzed by laser scanning densitometry (Personal Densitometer; GE Healthcare).
7
Quantitative Analysis of Retinal Protein Expression
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Total retinal tissue lysates (n=6) were prepared by the addition of 500 µl sodium dodecyl sulfate (SDS) buffer [250 mM Tris (pH 6.8), 10% SDS, 500 mM dithiothreitol, 50% glycerol, 0.5% bromophenol blue and a 1:100 protease inhibitor cocktail (cat. no. P8340; Sigma-Aldrich)]. Total tissue extracts (20 µg) were separated on a 12% SDS polyacrylamide gel. Proteins were transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA) and blocked with 5% skim milk in TBST (TBS containing 0.05% Tween-20) at room temperature for 2 h. Subsequently, the membranes were hybridized at 4°C overnight in TBST with the primary antibodies: Anti-rPARP-1 (1:5,000; cat. no. 1051-1; Epitomics, Burlingame, CA, USA), and anti-rCaspase-3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Boston, MA, USA) overnight at 4°C. Following incubation with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibody, proteins were visualized with an enhanced chemiluminescence kit (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA). For normalization, blots were probed with β-actin, a housekeeping antibody (anti-β-actin; 1:1,000; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). The signal was detected by exposing X-ray films to the processed blots and analyzed by laser scanning densitometry (Personal Densitometer; GE Healthcare, Piscataway, NJ, USA).
8
Quantitative Protein Analysis in Cells
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Lysates from Hela cells (50 µg) and neurons (20 µg) were subjected to SDS-PAGE (14%) then transferred onto nitrocellulose filters (0.22 µm). Protein bands were detected by fluorescent SB, Mouse anti-c-Myc monoclonal 9E10 (Abcam), Mouse anti-VAMP2 monoclonal (Synaptic System) or Rabbit anti-GFP (Merck Millipore) primary antibody, the latter three followed by the respective peroxidase-conjugated secondary antibody (Biorad). Chemiluminescence was with Hyperfilm ECL (GE-Healthcare Bio-Sciences), signal acquired by Personal Densitometer (GE-Healthcare Bio-Sciences). For detection with SB, filters were incubated (5 nM SB-Alexa647 in blot solution, for 1 h at 24 °C) then washed (blot solution, five washes, 10 min each), and fluorescence detected by Typhoon800 (GE-Healthcare Bio-Sciences).
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