Cell pellets were collected using 0.25% trypsin in EDTA and stored at − 80 °C until used. They were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors and stored at − 80 °C until analysis by western blot. Protein concentration was measured by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) using the manufacturer’s protocol. The lysate was electrophoresed in SDS-PAGE and then transferred onto nitrocellulose membranes (GE Healthcare, Chicago, IL). After transfer, the membrane was incubated in freshly prepared 5% non-fat dry milk (BioRad, Hercules, CA) for 1 h and then incubated against the primary antibodies listed in Additional file 2 : Table S1a, diluted in 5% non-fat dry milk, overnight at 4 °C. The membranes were then incubated in secondary antibodies, listed in Additional file 2 : Table S1b, and then developed using the ECL method (GE Healthcare) and visualized using ChemiDoc MP image visualizer (BioRad). Quantification was performed using Image J.
Non fat dry milk
Manufactured by Bio-Rad
Sourced in United States, Germany, Italy, United Kingdom
Non-fat dry milk is a powdered form of skim milk that has been dehydrated. It is used as a base material in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (26 mentions)
Pvdf membrane, by Bio-Rad (18 mentions)
Bovine serum albumin (bsa), by Merck Group (14 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (14 mentions)
Tween 20, by Merck Group (14 mentions)
Nitrocellulose membrane, by Bio-Rad (13 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (11 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Trans blot turbo transfer system, by Bio-Rad (9 mentions)
Protease inhibitor cocktail, by Merck Group (9 mentions)
Chemidoc mp imaging system, by Bio-Rad (8 mentions)
Ripa buffer, by Merck Group (8 mentions)
Image lab software, by Bio-Rad (8 mentions)
Pvdf membrane, by Thermo Fisher Scientific (8 mentions)
Triton x 100, by Merck Group (8 mentions)
Protease inhibitor, by Merck Group (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
β actin, by Merck Group (7 mentions)
Laemmli sample buffer, by Bio-Rad (7 mentions)
305 protocols using non fat dry milk
1
Western Blot Protocol for Protein Analysis
2
Serum Corticosterone and Prolactin Levels in Rats
3
Western Blot Analysis of RNA Sensing Proteins
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Cells were washed with PBS and lysed with RIPA (Sigma) including 1× HALT protease inhibitor (Life Technologies) for 30 min on ice. Lysates were then cleared by centrifugation at 20,000 rcf for 10 min at 4°C, and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). 10–20 µg of protein was separated on a 12–4% Bis-Tris or 20–4% Tris-Glycine polyacrylamide gel. Proteins were transferred to nitrocellulose membranes by standard wet transfer and then briefly washed in Tris-buffered saline (TBS) and blocked in 3% nonfat dry milk (Bio-Rad) in TBS for 1–2 h. The membranes were then washed and incubated with primary antibodies (listed below) in 1% nonfat dry milk in TBS + 0.1% Tween 20 (TBS-T) under shaking at 4°C overnight. Membranes were washed with TBS-T and incubated with secondary antibodies for 1–2 h. Membranes were washed 2× with TBS-T and 2× with TBS, and then fluorescence was detected using a Li-COR Odyssey Fc instrument. Primary antibodies were anti-ADAR: Cell Signaling Technologies 14175; anti-hnRNPC: abcam ab10294; anti-GAPDH: Cell Signaling Technologies 5174; anti–RIG-I: Adipogen AG-20B-0009-C100; anti-MDA5: Enzo Life Sciences ALX-210-935-C100; and anti-UPF1: abcam ab109363; secondary antibodies were Li-COR goat–anti-rabbit IgG or goat–anti-mouse IgG, IRDye 680 RD or IRDye 800 CW conjugates.
4
Western Blot Analysis Protocol
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After samples were run on 4–20% gels, they were transferred to nitrocellulose membranes. Membranes were blocked in 5% (w/v) nonfat dry milk (Bio-Rad, Hercules, CA, USA) in TRIS-buffered saline (TBS) with 0.1% (v/v) Tween-20 (TBST) and then incubated with primary antibodies in 1% (w/v) nonfat dry milk. After washing with TBST, membranes were incubated with horseradish peroxidase-linked secondary antibodies. Membranes were washed with TBST and then TBS before incubation with Western Lightning Plus (PerkinElmer, Waltham, MA, USA) enhanced chemiluminescence reagent, followed by CCD imaging (iBright CL1000, ThermoFisher Scientific). The primary antibody against GAPDH (Cell Signaling Technology Cat 8884, RRID:AB11129865) was conjugated to horseradish peroxidase, so there was not a need for incubation with secondary antibodies.
5
Western Blot Analysis Protocol
6
Protein extraction and Western blot analysis
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LAPC4 cells were harvested and total cellular proteins were extracted using a lysis buffer (62.5 mM Tris-HCl pH 6.8, 100 mM dithiothreitol, 2% SDS, 10% glycerol). The cytosolic, nucleus and mitochondrial protein extracts were prepared as described (14 (link)). The protein concentrations were determined using the Bio-Rad Protein assay (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer's instructions. Protein (25 µg) was loaded in each lane, electrophoresed on a 15% SDS-PAGE, and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The blots were blocked with TBST buffer [500 mM NaCl, 20 mM Tris-HCl (pH 7.4), and 0.1% Tween-20] containing 5% nonfat dry milk (Bio-Rad Laboratories) and then incubated with specific primary antibody in TBST buffer containing 1% nonfat dry milk at 4°C overnight. Following secondary antibody incubation for 1 h at room temperature, bands were visualized using a Super Signal Chemiluminescence kit (Millipore, Billerica, MA, USA), and exposed to Kodak X-Max film. β-actin was used as the internal control after stripping off the original membrane. The images were scanned and relative band intensities were calculated using the NIH ImageJ software.
7
Immunoblotting of LASP1 Protein
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Cells were lysed in Laemmli-buffer containing 10% β-mercaptoethanol (Sigma-Aldrich). Equal amounts of cells were resolved by 10% SDS-PAGE. After blotting on a nitrocellulose membrane (Schleicher&Schuell, Dassel, Germany) the membrane was blocked with 3% nonfat dry milk (Biorad) in TBS-T buffer (10 mM TRIS, 150 mM NaCl, 0.1% (w/v) Tween, pH 7.5). Then the membrane was incubated with a self-generated primary antibody against LASP1 [35 (link)] diluted 1:8000 and anti-β-Actin by Santa Cruz (Santa Cruz, CA, USA) diluted 1:2000. Finally the membrane was washed with TBS-T and incubated with the secondary antibody goat-anti-rabbit horseradish peroxidase-coupled and diluted 1:5000 (Biorad). The amount of detected protein was visualized by enhanced chemiluminescence (Amersham Biosciences, Freiburg, Germany) and autoradiography. Quantification of autoradiography signals was carried out by densitometry using the ImageJ software (NIH, Bethesda, USA).
8
Quantification of CV-N Binding to Recombinant HIV-1 gp120
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Recombinant HIV‐1IIIB gp120 (100 ng/well, ImmunoDiagnostics, Woburn, MA) coated on a 96‐well plate, was blocked with 3% nonfat dry milk (Bio‐Rad) for 2 h and washed thrice with PBST before being incubated with serial dilutions of rCV‐N or control CV‐N (produced in E. coli) for 2 h. Wells were washed with PBST, incubated for 1 h with a 1:1000 dilution of polyclonal rabbit anti‐CV‐N antibody (NIH) and washed as before. Wells were incubated with peroxidase‐conjugated goat anti‐rabbit secondary antibody (Thermo Fisher Scientific Inc., Rockville, MD) at a 1 : 1000 dilution for 1 h. After washing, detection was carried out with the TMB 2‐component Microwell peroxidase substrate kit (KPL Inc., Gaithersburg, MD) according to the manufacturer's protocol, and absorbance was read at 450 nm.
9
Recombinant gp120 Protein Binding Assay
10
SARS-CoV-2 Antibody Neutralization ELISA
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High-protein binding 96-well ELISA plates (Corning) coated with PBS alone or with 2 μg/ml of SARS-CoV-2 S protein in PBS overnight at 4°C were blocked for 1 hour with 3% nonfat dry milk (Biorad) in PBS. Serum samples diluted in DMEM (1:50 dilution) were serially incubated 4 times for 1 h each at 37°C on S protein-coated or control wells. The depleted sera were tested for their neutralization capacity as described above.
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