HeLa 229 cells (ATCC, CCL-2.1, USA) were transfected with either pcDNA3 or pcDNA3/FlaB3 using the LipofectamineTM 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Transfected HeLa cells were harvested and detected using immunoblotting analysis with antibodies against FlaB3. The primary anti-FlaB3 antibodies were provided by the Institute of Pathogen Biology at the University of South China. The secondary antibody was horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (GeneTex, San Antonio, TX, USA). Finally, the ECL Prime Western Blotting Detection Reagent (Thermo Fisher Scientific, Fremont, CA, USA) was used as recommended to visualize the protein bands with a G:BOX Chemi XX9 (Syngene, Cambridge, UK) digital imager.
Ecl prime western blotting detection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
ECL Prime Western Blotting Detection Reagent is a chemiluminescent substrate for the detection of proteins on Western blots. It is designed to provide sensitive and stable signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Bolt mini gel tank, by Thermo Fisher Scientific (2 mentions)
Western lightning plus ecl, by PerkinElmer (2 mentions)
Precision plus protein standard, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
G box chemi xx9, by Syngene (1 mentions)
Lipofectaminetm 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Streptavidin protein, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Mops running buffer, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Amersham imager 600 system, by GE Healthcare (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Protease, by Roche (1 mentions)
10 protocols using ecl prime western blotting detection reagent
1
Transfection and Detection of FlaB3 Protein
2
Western Blot Analysis of Apolipoproteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE was performed using 18% polyacrylamide gel under reducing condition. The separated proteins were transferred to PVDF membranes (Merck Millipore). After blocking the membrane with 5% skim milk, apoC-II, apoC-III, or apoA-I was primarily detected with goat anti-apoC-II, -apoC-III, or -apoA-I polyclonal antibody (Academy Bio-Medical Company), respectively, and horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody (Medical & Biological Laboratories) was the secondary antibody. Finally, the band containing apoC-II or apoC-III was visualized using ECL Prime Western Blotting Detection Reagent (GE Healthcare), and the apoA-I band was visualized using 3,3′-diaminobenzidine tetrahydrochloride and hydrogen peroxide. In the case of biotinylated proteins, the detection was performed similarly except that 5% BSA was used as a blocking solution and detection was carried out using Streptavidin Protein, HRP (Thermo Fisher scientific), and ECL Prime Western Blotting Detection Reagent.
3
Western Blot Analysis of Ago1 and FLAG
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were resolved on a polyacrylamide gel (NuPAGE Bis-Tris 4%–12% gel, Life Technologies) using a Bolt Mini Gel Tank (Thermo Fisher Scientific) and MOPS running buffer (Life Technologies) as per the manufacturer's protocol. Proteins were then transferred to a PVDF membrane (Life Technologies) in transfer buffer (Life Technologies) using the Bolt Mini Gel Tank, according to the manufacturer's protocol. Membranes were blocked by incubation with 5% milk in PBST for 1 h at 4°C, and then incubated overnight at 4°C with primary antibody diluted in 5% milk in PBST. The following day, blots were washed with PBST for 5 min four times, incubated for 1 h shaking at room temperature with secondary antibody diluted in PBST, and then washed again with PBST for 5 min four times. Blots were visualized using the ECL Prime Western Blotting Detection Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol, and imaged using a Bio-Rad ChemiDoc MP.
For primary antibodies, dilutions were 1:1000 for anti-Ago1 (Abcam, ab5070) and 1:2000 for anti-FLAG (Sigma Aldrich, #F1804). For secondary antibodies, dilutions were 1:10,000 for both HRP-conjugated anti-mouse and anti-rat IgG (Sigma Aldrich).
For primary antibodies, dilutions were 1:1000 for anti-Ago1 (Abcam, ab5070) and 1:2000 for anti-FLAG (Sigma Aldrich, #F1804). For secondary antibodies, dilutions were 1:10,000 for both HRP-conjugated anti-mouse and anti-rat IgG (Sigma Aldrich).
4
Western Blot Analysis of HSA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins from patients' serum were separated by SDS‐PAGE, as previously described, and transferred by the Semi‐Dry method to nitrocellulose membranes (Bio‐Rad). Then, HSA detection was performed by incubating the membrane with an anti‐HSA primary antibody, which was produced following the technique described by Albar et al. in 1981.29 Subsequently, an appropriate goat anti‐rabbit HRP‐conjugated secondary antibody (Jackson Laboratory) was added. Finally, proteins were visualized using the chemiluminescent ECL Prime Western Blotting Detection Reagent (Thermo Scientific) and the Amersham Imager 600 system (GE Healthcare). The quantification of the bands was carried out with the ImageJ software.30
5
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were scrapped and homogenized in medium-salt lysis buffer (150 mM NaCl, 50 mM Tris pH 8, 1% NP40 and protease inhibitors cocktail) and concentrations determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher). Lysates were loaded in acrylamide gels for electrophoresis in Tris-Glycine SDS Running Buffer. Primary antibodies were incubated overnight at 4 °C. Secondary HRP-conjugated antibodies were incubated the following day for 1 h at room temperature, and ECL Prime Western Blotting Detection Reagent (Fisher Scientific) was used as a chemiluminescent reagent for protein detection. Antibodies and dilutions are listed in Supplementary Table 7 .
6
Western Blot Analysis of Protein Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tissues were homogenized in 2% SDS lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2% SDS) supplemented with protease (Roche) and phophatase (Sigma-Aldrich) inhibitors cocktails. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher). Lysates were loaded in 12% bis-tris acrylamide gels and transferred to nitrocellulose membranes. Primary antibodies were incubated overnight at 4°C. Secondary HRP-conjugated antibodies were incubated the following day for 1 h at room temperature, and ECL Prime Western Blotting Detection Reagent (Fisher Scientific) or SuperSignal™ West Dura Extended Duration Substrate were used as a chemiluminescent reagent for protein detection. Antibodies and dilutions are listed in Supplementary Table S2 .
7
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell cultures were harvested and centrifuged at 200 × g for 15 min. Cell pellets were lysed in RIPA buffer (Cell signaling) for analysis of SREBP2, SREBP1 and MES buffer (Jiang et al., 2013b (link)) for analysis of other proteins. All lysis buffers were supplemented with phosphatase inhibitors. Upon lysis cell lystates were mixed with Tricine-SDS sample buffer (Invitrogen) and 2% β-mercaptoethanol, boiled for 5 min and resolved by SDS-PAGE using 10–20% Tris/Tricine gel (Bio-rad). Precision plus protein standards (Bio-Rad) were include in SDS-PAGE as reference markers. After gel elctrophoresis, proteins were transferred onto nitrocellulose for further probing with antibodies against proteins of interest. Western Lightning Plus ECL (PerkinElmer) or ECL™ Prime Western Blotting Detection Reagent (Fisher Scientific) were used for visualization of protein immunoreactivities. Immunoreactivity of β-actin was used as loading controls.
8
Western Blot Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in 200–500 μL of RIPA buffer (Thermo Fisher Scientific 89901) containing protein inhibitors cOmplete, Mini, EDTA-free protease inhibitor cocktail (Millipore Sigma 11836170001). Subsequently, samples were sonicated twice for 5 sec (power set to 50%) while samples were on ice. Cleared supernatant from lysates were recovered by centrifugation and the protein was quantified using Pierce BCA protein assay kit (Thermo Scientific 23225) according to the manufacture's protocol. Protein (15 or 20 μg) was loaded into 6%, 10%, or 12% SDS-polyacrylamide gels and, subsequently, transferred onto a PVDF membrane in a semi-dry apparatus according to standard Western Blot protocols. Membranes were blocked and later they were incubated with primary antibodies overnight. We used GAPDH antibody (1:6000 dilution; Cell Signaling 5174S) or β-tubulin (1:1000 dilution; Cell Signaling 2128S) as a normalizer for protein expression. The other primary antibodies used are the following: FLAG (1:1000 dilution; Sigma F1804), p53 (DO-I) (1:1000 dilution; Santa Cruz Biotechnology sc-126), CD44 (1:1000; dilution Cell Signaling, Catalog no. 3570S), mouse IgG (Santa Cruz Biotechnology sc-2025) and ZMAT3 (1:250 dilution), that was generated from Abgent. Membranes were developed using ECL Prime Western blotting detection reagent (Fisher Scientific RPN2232).
9
Whole Cell Lysate Preparation and Western Blotting
10
Optimized Intracellular Protein Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To collect total intracellular protein samples, cells were lysed in RIPA buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0), supplemented with Complete Protease Inhibitor Cocktail (Roche) and frozen at −80 °C. Cellular debris was pelleted by centrifugation at 16,000× g for 30 min at 4 °C, and the supernatant was quantified by BCA Protein Assay (ThermoScientific, Waltham, MA, USA). Ten micrograms of sample were loaded onto 10–12% SDS-PAGE gels. Samples were transferred onto Immobilon-P PVDF membranes (Millipore), blocked in 5% milk, and incubated overnight with primary antibodies diluted in 5% BSA: rabbit anti-PCBP1 (clone EPR11055, Abcam ab168378, diluted 1:10,000); rabbit anti-actin (A2066, Sigma, St. Louis, MO, USA, 1:20,000); mouse anti-HCV core (clone B2, Anogen MO-I40015B, 1:7500); mouse anti-JFH-1 NS5A (clone 7B5, BioFront Technologies, Tallahassee, FL, USA, 1:10,000). Blots were incubated for 1 h with HRP-conjugated secondary antibodies diluted in 5% skim milk: anti-mouse (HAF007, R&D Systems, Minneapolis, MN, USA, 1:25,000); anti-rabbit (111–035-144, Jackson ImmunoResearch Laboratories, West Grove, PA, USA, 1:50,000) and visualized using enhanced chemiluminescence (ECL Prime Western Blotting Detection Reagent, Fisher Scientific).
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!