Validation of mRNA (CUL2 and RBBP4) expression at the protein level was performed by western blot. MDMs infected with HIV-1/HIV-2 and uninfected controls, cultured for 7 days post infection were collected by scrapping followed by centrifugation. Proteins were extracted with RIPA buffer (Cat # 89901, Thermo Scientific) supplemented with protease inhibitor cocktail (Cat # 4693159001, Sigma-Aldrich). Equal amount of proteins (5 µg) were used in the Western blot experiment as described earlier28 (link) with m CUL 2 monoclonal antibody (Santa Cruz Biotechnology, Inc., Cat # sc-166506)67 (link), RBBP4 monoclonal antibody (Santa Cruz Biotechnology, Inc., Cat # sc-373873)68 (link) and β-Actin monoclonal antibody (Santa Cruz Biotechnology, Inc., Cat # sc-47778)69 (link). ECL western blotting analysis system (Cat # RPN2108, GE Healthcare) was used to visualize the proteins.
Ecl western blotting analysis system
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Germany, Italy
The ECL Western Blotting Analysis System is a laboratory equipment product that enables the detection and quantification of proteins in biological samples using the Western blotting technique. The system provides the necessary components and reagents for the chemiluminescent detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Cell lysis buffer, by Cell Signaling Technology (8 mentions)
Hybond, by Cytiva (6 mentions)
Immobilon p membrane, by Merck Group (6 mentions)
Anti β actin monoclonal antibody, by Merck Group (5 mentions)
Nitrocellulose membrane, by Cytiva (5 mentions)
Luminata crescendo western hrp substrate, by Merck Group (5 mentions)
Amersham imager 600, by GE Healthcare (4 mentions)
Amersham imager, by Cytiva (4 mentions)
Las 3000 imager, by Fujifilm (4 mentions)
Complete mini, by Roche (4 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (4 mentions)
Membrane blocking solution, by Thermo Fisher Scientific (4 mentions)
0.45 μm nitrocellulose membrane, by Bio-Rad (4 mentions)
Mini protean 3 cell system, by Thermo Fisher Scientific (4 mentions)
Hrp conjugated α tubulin, by Cell Signaling Technology (4 mentions)
Phosphatase inhibitor, by Roche (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Mini protean tgc gel, by Bio-Rad (3 mentions)
Amersham ecl western blotting, by Cytiva (3 mentions)
Secondary antibodies against rabbit or mouse igg conjugated to horseradish peroxidase, by Cell Signaling Technology (3 mentions)
91 protocols using ecl western blotting analysis system
1
mRNA Expression Validation by Western Blot
2
Quantitative Analysis of PFK Isoforms
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Western blotting was performed using the cytosolic fraction. Samples were boiled at 95°C
for 5 min in SDS sample buffer (Red Loading Buffer, New England Biolabs, Ipswich, MA,
U.S.A.). Equal units (0.3 mU) of PFK-1 activity present in the unpurified cytosolic
protein samples obtained from various organs were loaded in each lane of a 7.5%
polyacrylamide gels (Mini-PROTEAN TGX gel, Bio-Rad Laboratories, Hercules, CA, U.S.A.) and
separated by electrophoresis. The separated proteins were then transferred to
polyvinylidene difluoride membranes, and treated with blocking reagent (Block Ace, DS
Pharma Biomedical, Osaka, Japan) for 50 min. Thereafter, the membranes were incubated for
120 min with primary rabbit monoclonal antibodies (Abcam, Cambridge, U.K.) against PFK-M
(ab154804, 1:1,000), PFK-L (ab181064, 1:500), PFK-P (ab204131, 1:1,000), and FBP-1
(ab109732, 1:1,000). After washing, the membranes were incubated with ECL Anti-Rabbit IgG,
Horseradish Peroxidase Linked Whole Antibody from donkey (GE Healthcare, 1:10,000) for 90
min. Immunoreactivity was detected with chemiluminescence (ECL Western blotting Analysis
System, GE Healthcare). The chemiluminescent signals of the membranes were measured, and
densitometric analyses were performed using a chilled CCD camera (ImageQuant LAS 4000
mini, GE Healthcare).
for 5 min in SDS sample buffer (Red Loading Buffer, New England Biolabs, Ipswich, MA,
U.S.A.). Equal units (0.3 mU) of PFK-1 activity present in the unpurified cytosolic
protein samples obtained from various organs were loaded in each lane of a 7.5%
polyacrylamide gels (Mini-PROTEAN TGX gel, Bio-Rad Laboratories, Hercules, CA, U.S.A.) and
separated by electrophoresis. The separated proteins were then transferred to
polyvinylidene difluoride membranes, and treated with blocking reagent (Block Ace, DS
Pharma Biomedical, Osaka, Japan) for 50 min. Thereafter, the membranes were incubated for
120 min with primary rabbit monoclonal antibodies (Abcam, Cambridge, U.K.) against PFK-M
(ab154804, 1:1,000), PFK-L (ab181064, 1:500), PFK-P (ab204131, 1:1,000), and FBP-1
(ab109732, 1:1,000). After washing, the membranes were incubated with ECL Anti-Rabbit IgG,
Horseradish Peroxidase Linked Whole Antibody from donkey (GE Healthcare, 1:10,000) for 90
min. Immunoreactivity was detected with chemiluminescence (ECL Western blotting Analysis
System, GE Healthcare). The chemiluminescent signals of the membranes were measured, and
densitometric analyses were performed using a chilled CCD camera (ImageQuant LAS 4000
mini, GE Healthcare).
3
Cranial Mesenchyme Protein Profiling
4
Cell Culture Reagents and Antibodies
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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin, L-glutamine were obtained from GIBCO (Paisley, Scotland). DNase 1, gelatin, lypopolysaccharide (LPS), 1,10 phenanthroline (PA), poly-L-lysine (PLL), trypsin, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), phorbol 12-myristate 13-acetate, bovine serum albumine was provided by Sigma (St. Louis, MO, USA). Glial fibrillary acidic protein (GFAP) antibodies were purchased from Serotec (Oxford, UK). Standard proteins, G-250and R-250 coomassie brilliant blue were purchased from Bio-Rad (Hercules, CA, USA). Purified MMP-2 and MMP-9 were purchased from Alexis Biochemicals (San Diego, CA, USA). Primer pairs specific for MMP-2, MMP-9 and 18S were from Sigma Genosys (Cambridge, UK). RNeasy mini kit and QuantiTect Reverse Transcription were from Qiagen (Valencia, CA, USA). Antibodies against extracellular–regulated protein kinases (ERK) 1/2, and phosporilated ERK 1/2 (p-ERK 1/2) were from Santa Cruz Biotechnology (Santa Cruz, CA). Hybond-P PVDF membranes, enhanced chemiluminescence (ECL) Western Blotting Analysis System and anti-mouse-HRP secondary antibody were from GE Healthcare Life Sciences (Little Chalfont, Buckinghamshire, UK).
5
Western Blot Analysis of Pb18Cyb5 Protein
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Proteins in SDS-PAGE were transferred to the nitrocellulose membrane that was then incubated with polyclonal anti-Pb18Cyb5 at 1:250 dilution for 2 h at room temperature. After washing, the membranes were incubated with peroxidase-coupled mouse anti-IgG secondary antibody (1:1,000 dilution). The reaction was revealed by chemiluminescence with the ECL Western Blotting Analysis System (GE Healthcare). Negative control was obtained with pre-immune mouse serum (1:250 dilution). Reaction was developed in a chemiluminescent imager (Amersham Imager 600, GE Healthcare).
6
Quantitative Western Blot Analysis
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Extracted nuclear protein was denatured in SDS loading buffer and separated by SDS-PAGE (5%-12% acrylamide gradient gels), then transferred onto Amersham Hybond 0.2 PVDF blotting membrane (GE Healthcare Life Sciences, USA). Rabbit anti-NF-κBp65 monoclonal antibody or rabbit anti-β-actin monoclonal antibody (Cell Signaling Technology, Massachusetts, USA) was applied to the membrane for 1 h at 37°C after blocking with 5% nonfat milk. This was followed by incubation with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Cell Signaling Technology, Massachusetts, USA). Finally, the proteins were visualized by a chemiluminescence ECL western blotting analysis system (GE Healthcare, Piscataway, NJ, USA). The protein levels were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and were normalized to β-actin.
7
Western Blot Protein Analysis
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Samples were resolved using NuPAGE Novex Bis-Tris pre-cast gels (Life Technologies, 12 % 10-well NP0341BOX) using the BLUeye Pre-Stained Protein Ladder (Geneflow Ltd, S6-0024) as a protein size reference. Proteins were transferred onto Immobolin PVDF membranes (Millipore, IPFL00010) and protein gel blot analysis was performed using indicated antibodies and visualized on Hyperfilm ECL (GE Healthcare, 28-9068) using the ECL western blotting analysis system (GE Healthcare, RPN2109). Adobe Photoshop software was used to crop full blots and band integrated densities were quantified using the ImageJ software.
8
Western Blot Analysis of Mitochondrial Dynamics
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Western blot analysis was performed as previously reported48 (link). Briefly, the cells were lysed with Cell Lysis Buffer (Cell Signaling Technology). The proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). The membranes were probed with anti-Drp1 monoclonal antibodies (1:1000; Cell Signaling Technology), anti-Fis1 polyclonal antibodies (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Mfn1 polyclonal antibodies (1:1000; Cell Signaling Technology), anti-Mfn2 monoclonal antibodies (1:1000; Cell Signaling Technology), anti-Opa1 monoclonal antibodies (1:1000; BD Biosciences), anti-ERK1/2 polyclonal antibodies (1:1000; Cell Signaling Technology), anti-phospho ERK1/2 (Thr202/Tyr204) monoclonal antibodies (1:2000; BD Biosciences), and anti-β-actin monoclonal antibodies (1:5000; Sigma-Aldrich). The membranes were then incubated with secondary antibodies against rabbit or mouse IgG conjugated to horseradish peroxidase (Cell Signaling Technology). The bands were visualized using an ECL Western Blotting Analysis System (GE Healthcare, Buckinghamshire, UK). Images were acquired using an LAS-3000 Imager (FUJIFILM, Tokyo, Japan).
9
Western Blotting Procedure for Protein Analysis
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Western blotting was performed as described previously.10 Cells were lysed on ice for 20 minutes in SDS‐modified RIPA buffer containing protease and phosphatase inhibitor cocktails (cOmplete Mini and PhosSTOP EASYpack, respectively; Roche Diagnostics, Mannheim, Germany) and then centrifuged at 12,000 g at 4°C for 20 minutes. The resulting cell lysates (20 µg samples) were boiled with Laemmli sample buffer and subjected to SDS‐PAGE. The samples were transferred to a polyvinylidene difluoride membrane (Millipore), which was blocked for 1 hour in Block Ace (DS Pharma, Osaka, Japan) at RT, and then incubated overnight at 4°C with primary antibodies against pS6 (1:2000, S235/S236), S6 (1:1000, Cell Signaling Technology), and GAPDH (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with 1 × TBS containing 0.1% Tween 20, membranes were incubated for 1 hour at RT with appropriate secondary antibodies, followed by rewashing. Finally, the signals were detected using an ECL Western blotting analysis system (GE Healthcare, Piscataway, NJ, USA) in accordance with the manufacturer's instructions.
10
Characterization of Poly-P Metabolism Genes
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The fragments of promoters of genes involved in poly-P metabolism were amplified by PCR using primers listed in Table S1 . The PCR products were labeled by biotin at the 3′-end using the Biotin 3′ End DNA Labeling kit (Thermo Scientific). Purified His-tagged PolR was used in the assays, with described procedures (Zhang et al., 2014 (link)). For competition assays, excess of unlabelled specific probes and non-specific probes were added into the reaction. The mixtures were analyzed on non-denaturing page (8% polyacrylamide), then the DNA probes were transferred to nylon membrane and developed by ECL Western blotting Analysis System (GE Healthcare).
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