Phosphate-buffered saline was flushed through both the upper and lower channels and total protein was extracted in situ using cell lysis buffer supplemented with protease and phosphatase inhibitor (# MSSAFE, Sigma, Carlsbad, CA). Protein concentration was then determined and equal amounts of protein lysates were heat denatured and separated on a 10% mini-protean precast gel (Bio-Rad, Hercules, CA). Gel was then transferred on to midi format 0.2 μm Nitrocellulose Membrane (Bio-Rad). Blot was blocked in Odyssey Blocking Buffer (LI-COR, Lincoln, NE) for 1 hour and then incubated in primary antibody for STAT1, pSTAT1, and glyceraldehyde-3-phosphate dehydrogenase (Supplementary Table 1 ) overnight at 4ºC. After incubation with infrared conjugated secondary antibodies (LI-COR) for 1 hour, blots were washed with TBST and bands were visualized by scanning membrane on Odyssey Infrared Imaging System (Li-COR). Image J analysis was used to quantify levels of phosphorylated STAT1 compared with glyceraldehyde-3-phosphate dehydrogenase.
Ms safe
Manufactured by Merck Group
Sourced in United States
MS-SAFE is a laboratory equipment product designed to provide a secure environment for mass spectrometry analysis. It is a containment system that ensures the safety and protection of users and samples during the mass spectrometry process.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Odyssey blocking buffer, by LI COR (4 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Infrared conjugated secondary antibodies, by LI COR (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Bca analysis, by Thermo Fisher Scientific (2 mentions)
Mini protean precast gel, by Bio-Rad (1 mentions)
Image lab v6, by Bio-Rad (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Gel doc system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
13 protocols using ms safe
1
Western Blot Analysis of STAT1 Activation
2
Detection of BRCA1 Protein in iPSCs
3
Striatal Protein Expression Analysis
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Striatal tissue samples were lysed in RIPA buffer with protease and phosphatase inhibitors (MSSAFE, Sigma), centrifuged at 16,000 x g and the soluble fraction collected for subsequent protein analysis. Total protein content was determined by BCA analysis (ThermoFisher Scientific) and 20 ug of protein resolved on a 10% Tris-Glycine gel by electrophoresis (BioRad, Hercules, CA). Total protein was transferred to nitrocellulose membranes (BioRad), blocked (Genesee Scientific) and probed with the following antibodies overnight at 4 °C: Tyrosine hydroxylase (MAB318, Millipore Scientific, Burlington, MA) and mouse anti-beta actin (1:5000, LI-COR, Cat# 926-42212, RRID: AB_2756372). Membranes were washed, incubated with corresponding goat anti-mouse or goat anti-rabbit conjugated near-infrared secondary fluorescent antibodies (1:5000, LI-COR, Cat# 926-32211, RRID: AB_621843; and Cat# 926-68070, RRID: AB_10956588), and scanned on an Odyssey imaging system (Licor, Lincoln, Nebraska, USA). Relative protein expression was quantified by optical density and normalized to beta-actin as loading control using the Licor imaging program.
4
Quantifying STAT1 and pSTAT1 Levels
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HIOs were washed with 1 PBS and total protein was extracted in situ using a cell lysis buffer supplemented with protease and phosphatase inhibitor (# MSSAFE, Sigma-Aldrich, Carlsbad, CA, USA). Protein concentration was then determined, and equal amounts of protein lysates were heat-denatured and separated on a 10% mini-protean precast gel (Bio-Rad, Hercules, CA, USA). Gel was then transferred on to midi format 0.2 µm Nitrocellulose membrane (Bio-Rad). Blot was blocked in Odyssey Blocking Buffer (LI-COR, Lincoln, NE, USA) for 1 h and then incubated in primary antibody (Table 2 ) for STAT1, pSTAT1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) overnight at 4 °C. Blots were washed three times for 10 min each in 1× tris-buffered saline + 0.1% Tween-20 (TBST). After incubation with infrared conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) for 1 h, blots were washed with TBST and bands were visualized by scanning membrane on Odyssey Infrared Imaging System (Li-COR Biosciences).
5
Immunoblotting Experiment Protocol
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For all immunoblotting experiments, cells were grown in six-well plates and used at 75% confluency for treatments. After the indicated treatments, the cells were washed with ice-cold PBS and lysed using RIPA lysis buffer, supplemented with protease–phosphatase inhibitor (MS-SAFE; Sigma-Aldrich). Protein concentration was determined using Bio-Rad DC protein assay (Bio-Rad laboratories). 30 μg cellular protein lysates were resolved by 12% SDS–PAGE, transferred to PVDF membrane (Bio-Rad), and probed with antigen-specific primary antibodies. Blocking was performed with 5% skim milk; except for the phospho antibodies, which was instead performed with 5% bovine serum albumin. For all analyses, HRP-conjugated secondary antibodies were used (peroxidase-conjugated goat anti-rabbit IgG or peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch)) and detection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). The results were analyzed using Image Lab v6.1 by Bio-Rad laboratories.
6
Western Blot Analysis of Neutrophil Proteins
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Neutrophils were lysed in boiling RIPA lysis buffer (60 mM Tris, pH 7.5, 10% Glycerol, 1% TX-100, 150 mM NaCl, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM EDTA) and 1 X protease and phosphatase inhibitor (#MSSAFE, Sigma-Aldrich). Protein lysate (30 μg) with 2 X Laemmli buffer (in 1:1 ratio) was loaded onto a 4–20% gradient gel (Bio-Rad, Mississauga, ON, Canada). The gel was transferred onto a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 1 hr at room temperature in a blocking buffer (5% BSA in PBS-T), with a solution containing primary antibodies. After washing and the addition of the secondary antibody, membranes were developed using SuperSignal West Dura and photographed using the Bio-Rad GelDoc system with Image Lab software. The denistometric quantifications were performed using ImageJ software, version 1.51 v (Schindelin et al., 2012 (link)).
7
Detection of BRCA1 Protein in iPSCs
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iPSCs were treated with the proteasome inhibitor MG132 for 24 hours, and treated and nontreated cells were gently scraped off the plates, washed with phosphate buffered saline (PBS), and centrifuged at 15000 RPM for 1 min. Samples were lysed using 1X NETN buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5 mM EDTA and 0.5% NP-40) supplemented with phosphatase/protease inhibitor cocktail (MS-SAFE, Sigma-Aldrich). Lysates were sonicated at a frequency of 20 kHz for 5 s (3times) on ice and incubated on ice for 20 min. Samples were centrifuged for 20 min at 4°C at 15000 RPM. Total soluble protein concentrations were measured using a Bradford assay (BIO-RAD). 4X Laemmli sample buffer (BIO-RAD 161–0774) was added to 100 μg of total protein extracts and samples were boiled for 5 min. Samples were run in 4%–20% Mini-PROTEAN TGX Precast gels (BIO-RAD, 456–1094) and transferred to nitro-cellulose membrane using 1X transfer buffer (25mM Tris, 190 mM glycine, 20% Methanol and 0.1% SDS) over night at 4°C. Membranes were blocked with Odyssey blocking buffer (LI-COR) and then incubated with primary BRCA1 antibody (Millipore, OP92), which is raised against the N-terminal region of BRCA1 protein, overnight at 4°C. Following incubation with dye-labeled mouse secondary antibody for 2 hours at room temperature, signals were visualized using an Odyssey Fc imaging system (LI-COR).
8
Isolation of Rhipicephalus sanguineus Organs
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Unfed females of Rhipicephalus sanguineus were obtained from a tick colony as previously described (12 (link)). The ticks were externally rinsed in a mild detergent solution (1% Alconox, USA) for 1 min, surface sterilized in 1% sodium hypochlorite for 1 min, and finally washed three times in sterile saline. Dissections were performed on ice under a stereomicroscope using sterile forceps and microdissection scissors as previously described (5 (link)). Briefly, live ticks were anesthetized by chilling on ice, affixed on wax and covered with 50–150 μl of ice-cold phosphate-buffered saline (PBS, Biological Industries, Israel) containing protease inhibitors (MSsafe, Sigma, MO, USA). Organs were removed and washed in 100 μl of ice-cold PBS and immediately frozen (−80°C) in 25 μl of PBS. Care was taken to remove only the distal part of each Malpighian tubule containing CLEs (5 (link)). Ovaries were removed intact. Protein was extracted from 20 individual samples of Mt and Ov per sample as described next, in three biological replicates (Table 1 ).
9
Protein Extraction and Detection from Stored Heart Tissue
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Stored heart tissue was homogenized in RIPA buffer along with a protease inhibitor (MS-SAFE, Sigma-Aldrich, USA). Homogenate was then centrifuged at 10,000 rpm for 15 min and the protein concentration in the supernatant was estimated using Bradford’s assay. A total of 40 μg of proteins were separated on 12% denatured resolving gel through polyacrylamide gel electrophoresis (PAGE) in reducing condition and transferred to a nitrocellulose membrane (0.2 µm; Bio-rad, USA). Thereafter, the membrane was blocked with BSA (3%) for 2 h at RT. The membrane was incubated overnight with the respective primary antibodies along with the GAPDH antibody, followed by the particular secondary antibodies in a specific combination for 2 h at RT. After that, the bands having HRP-conjugated antibody were detected by the enhanced Chemiluminescence (ECL) method using ECL kit (Clarity western enhanced luminescence kit, Bio-rad, USA) while the fluorescence (PE)-labelled antibody was detected using MultiFluor Red channel with red epi light and Far Red (710 nm) filter under gel documentation system, FluorChem M, Protein Simple, California, USA.
10
EGFR Immunoprecipitation from CAL27 and HSC-3 Cells
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