Cytokines were measured using the Mouse Proinflammatory 7-Plex Ultra-Sensitive kit (Meso Scale Discovery). Serum IgM was measure by ELISA (eBioscience). To examine protein expression in tissues, lysates were run on SDS-PAGE and blotted onto nitrocellulose membranes (Invitrogen). Blots were probed for β-actin (Sigma), RIP1 (BD Biosciences) and RIP3 (Imgenex). For TNF activation experiments, BMDM were treated with 50ng/ml of TNF (R&D systems) and lysates were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Invitrogen). Blots were probed for IκB, phospho-IκB, tubulin, phospho-p38, total p38, phospho-JNK and total JNK (Cell Signaling), phospho-ERK and total ERK (Santa Cruz).
Nitrocellulose membrane
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, Germany, Switzerland, Sweden, Italy, Japan, Netherlands, Denmark
The Nitrocellulose membrane is a porous membrane made from cellulose treated with nitric acid. It is commonly used in various laboratory applications for the immobilization and detection of proteins, nucleic acids, and other biomolecules through techniques such as Western blotting, dot blotting, and nucleic acid hybridization.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (77 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (56 mentions)
Ripa buffer, by Thermo Fisher Scientific (49 mentions)
Protease inhibitor cocktail, by Roche (45 mentions)
Odyssey infrared imaging system, by LI COR (41 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (36 mentions)
Protease inhibitor cocktail, by Merck Group (35 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (34 mentions)
β actin, by Merck Group (34 mentions)
Odyssey blocking buffer, by LI COR (30 mentions)
Bis tris gel, by Thermo Fisher Scientific (30 mentions)
Ripa buffer, by Merck Group (29 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (29 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (27 mentions)
Odyssey imaging system, by LI COR (26 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (24 mentions)
Chemidoc mp imaging system, by Bio-Rad (24 mentions)
Gapdh, by Cell Signaling Technology (22 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (21 mentions)
β actin, by Santa Cruz Biotechnology (21 mentions)
1 311 protocols using nitrocellulose membrane
1
Cytokine and Signaling Pathway Analysis
2
Integrin-Mediated Signaling in Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For TF-Gold pull-down analysis, TF-Au, PEG-Au, and untreated endothelial cell pull-down isolates were resolved on NuPAGE 4-12% gradient Bis-Tris gels (Invitrogen), and transferred to a nitrocellulose membrane (Invitrogen). The membranes were probed with primary antibodies against Gal-3, α3, α4, α5, αV, β1, β3, β4, and β5 integrins, and Src kinase (see antibody list above) in conjunction with corresponding HRP-conjugated secondary antibodies and enhanced chemiluminescent (ECL) detection.
Endothelial cells from co-culture experiments (see below) were lysed using CelLytic M buffer with protein inhibitor cocktail (Sigma). Protein concentrations were determined using Protein Assay kit (Bio-Rad). Equal amounts of the protein from each sample (30 μg) were resolved on a NuPAGE 4-12% gradient Bis-Tris gels (Invitrogen), and transferred to a nitrocellulose membranes (Invitrogen). The membranes were sequentially probed with primary antibodies directed against phosphorylated and total Src, p38, MEK1/2, and Akt (see antibody list above) in conjunction with corresponding HRP-conjugated secondary antibodies and enhanced chemiluminescent (ECL) detection. Anti-β-Actin antibody (Abcam, Cambridge, CA) was used to control loading. The experiments were performed at least three times for each setting with same results.
Endothelial cells from co-culture experiments (see below) were lysed using CelLytic M buffer with protein inhibitor cocktail (Sigma). Protein concentrations were determined using Protein Assay kit (Bio-Rad). Equal amounts of the protein from each sample (30 μg) were resolved on a NuPAGE 4-12% gradient Bis-Tris gels (Invitrogen), and transferred to a nitrocellulose membranes (Invitrogen). The membranes were sequentially probed with primary antibodies directed against phosphorylated and total Src, p38, MEK1/2, and Akt (see antibody list above) in conjunction with corresponding HRP-conjugated secondary antibodies and enhanced chemiluminescent (ECL) detection. Anti-β-Actin antibody (Abcam, Cambridge, CA) was used to control loading. The experiments were performed at least three times for each setting with same results.
3
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were performed as previously described [29 (link)]. Briefly, immunoprecipitation of protein was performed by incubating 5 μg of antibody overnight in cell lysates. For these experiments, a standard buffer (150 mM NaCl, 20 mM Tris HCl (pH 7.5, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na3VO4, 1mg/ml leupeptin and 0.5 mM phenylmethylsulfonyl fluoride) was used (Cell Signaling). Protein A sepharose was then added in the lysate before centrifugation, and denaturation with NuPAGE sample-reducing agent (Invitrogen). Samples were then subjected to NuPAGE using 4–12% Novex Bis-Tris gels (Invitrogen) and separated proteins were transferred onto nitrocellulose membranes (Invitrogen). Protein was then revealed by western-blot. Samples were subjected to NuPAGE using 4–12% Novex Bis-Tris gels (Invitrogen) and proteins were transferred onto nitrocellulose membranes (Invitrogen). Membranes were probed with specific primary antibody (1/100) followed by secondary antibody coupled to peroxidase. Blots were revealed with the ECL substrate (Pierce).
4
PRRSV Viral-Like Particle Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TriEx Sf9 cells co-infected with recombinant baculoviruses containing PRRSV M, N, E, and GP5 gene were harvested at 72 h after infection. Cells were lysed with a lysis buffer containing 0.01 M Tris-HCl, 0.14 M NaCl, 0.025% NaN3, 1% Triton X-100, and protease/phosphatase inhibitors cocktail (Thermo Scientific, Rockford, IL). Cell lysates and purified VLPs were subjected to SDS-PAGE gel electrophoresis (Novex by Life Technologies, Carlsband, CA) and transferred to a nitrocellulose membrane (Life Technologies, Gaithersburg, MD). The membrane was then blocked for 1 h by rocking slowly at room temperature with 5% (w/v) milk powder in PBS plus 0.05% Tween 20 (PBST). Next, primary antibody, monoclonal mouse anti-HA antibody (Sigma-Aldrich, St. Louis, MO) diluted 1:5,000 in blocking buffer 5% (w/v) milk powder in PBST was added to the membrane and incubated overnight at 4 °C on the rocker. The secondary antibody, goat anti-mouse IRDye (LI-COR, Lincoln, NE) diluted 1:10,000 in PBST was added to the membrane and incubated for 1 h rotating at room temperature. Bands were visualized using the ODESSY Infrared Imaging System (LI-COR, Lincoln, NE).
5
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein was extracted from cells with ice-cold lysis buffer supplemented with protease inhibitor cocktail (Merck, Germany) and PMSF. The nuclear protein was extracted with Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China). The protein concentration was determined with Detergent Compatible Bradford Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated on SDS-PAGE gels and transferred onto nitrocellulose membrane (Life Technologies, USA). After blocking with Odyssey® blocking buffer (PBS) (LI-COR, Lincoln, USA), the membranes were incubated overnight with the primary antibodies of CD31 (ab28364,1:500), vWF (ab6994,1:500), α-SMA (ab32575,1:1000), vimentin (ab92547,1:1000), Histone H3 (ab1791,1:1000), β-catenin (ab32572,1:5000) and GAPDH (ab181602,1:10000) (abcam, Cambridge, UK). After that, IRDye 800CW Goat anti-Rabbit IgG (H + L) Secondary Antibody (926–32,211, 1:10000), IRDye® 680LT Goat anti-Rabbit IgG (H + L) Secondary Antibody (926–68,021) (LI-COR, Lincoln, USA) were added and incubated for 1 h at room temperature. Then the band intensities were scanned by Odyssey (LI-COR, Lincoln, USA) and normalized to GAPDH or Histone H3.
6
Western Blot Analysis of OVGP1 in Fallopian Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured human fallopian tissue was homogenized in ice-cold lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA) with proteinase inhibitor. Equal amounts of protein measured by the BCA protein assay kit (Thermo Scientific) were loaded into precast NuPAGE 4–12% gradient Bis-Tris. Gel electrophoresis was performed and proteins were dry-transferred using an iBlot system (Invitrogen) to a nitrocellulose membrane (Life Technologies). The blots were probed with a polyclonal anti-OVGP1 (1:1,000) antibody (LsBio) overnight at 4 °C, followed by anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5,000; Zymed). Proteins were detected by enhanced chemiluminescence primer (GE HealthCare Life Sciences) and exposed using a FluorChem HD machine (Alpha Innotech). The same blot was stripped with buffer (Thermo Scientific) and re-probed with a monoclonal anti-α-tubulin antibody (1:10,000; Sigma-Aldrich), followed by an anti-mouse secondary antibody conjugated to horseradish peroxidase.
7
Western Blot Analysis of Epithelial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were separated on 4–12% SDS-polyacrylamide gels (Life Technologies) and transferred to a nitrocellulose membrane (Life Technologies). Protein detection was performed using an anti-EpCAM monoclonal antibody (R&D Systems, Inc., Minneapolis, MN), anti-β-actin monoclonal antibody (Sigma-Aldrich), anti-E-cadherin monoclonal antibody (Cell Signaling Technology), anti-vimentin monoclonal antibody (Abcam), anti-phospho-Smad2 monoclonal antibody (Cell Signaling), and anti-Cyclin D1 monoclonal antibody (Cell Signaling).
8
M. smegmatis Protein Detection by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
M. smegmatis MC2155 strains with integrated FLAG-tagged constructs were grown in TSB with Tween overnight at 37 °C to an OD600 of ~1.0. Cells were harvested by centrifugation and resuspended in 1 x NuPage LDS sample buffer (Invitrogen) +5 mM sodium metabisulfite. Samples were heated at 95 °C for 10 min before loading onto a 4–12% gradient Bis-Tris mini-gel (Invitrogen). After separation, proteins were transferred to a nitrocellulose membrane (Life Technologies) or a PVDF membrane (Thermo Scientific). Membranes were probed with a monoclonal mouse anti-FLAG antibody (M2; Sigma). Secondary antibody and detection reagents were obtained from Lumigen (ECL plus kit) and used according to the manufacturer’s instructions.
9
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blotting, cells were resuspended in 2X Laemmli buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris-HCl pH 6.8) and boiled for 5 minutes. Samples were run on a Novex Tris-Glycine gel (Life Technologies) using FASTRun Buffer (Fisher Scientific). Samples were then transferred to a nitrocellulose membrane (Life Technologies). Membranes were rinsed twice with Tris-buffered saline (TBS) with 0.1% Tween-20 (Tw) and then blocked with 5% bovine serum albumin (BSA) in TBS/Tw. Membranes were incubated with primary antibody diluted in 0.5% BSA in TBS/Tw overnight at 4°C. Membranes were then washed 6 times with TBS/Tw and incubated with HRP conjugated secondary antibodies in TBS/Tw for 1 hour at room temperature. Signals were then developed using an enhanced chemiluminescence kit (Thermo Fisher) for 5 minutes and the signals were captured by UVP digital imaging system.
10
Investigating Calmodulin-TCF4 Interaction
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!