Protein samples were resolved using 4–12% or 10% NuPAGE Bis-Tris gels with NuPAGE MOPS SDS running buffer (Novex, Life Technologies), transferred to polyvinylidene difluoride (PVDF) membranes and blocked in 5% milk. Membranes were incubated with primary antibody diluted in phosphate buffered saline (PBS) with 5% milk and 0.2% Tween 20 at 4 °C overnight or 1 h at room temperature. Primary antibodies were as follows: 1 μg ml−1 mouse anti-FLAG mAb M2 (Sigma) for 1xFLAG and 3xFLAG tagged viral protein expression; 1:1,000 monoclonal anti-Vif (#316)78 (link). Proteins were detected with the ECL Plus Chemiluminescent System (Amersham Biosciences), Immobilon Western HRP Chemiluminescent Substrate (Millipore) or SuperSignal West Pico Chemiluminescence System (Thermo Scientific).
Ecl plus chemiluminescent system
Manufactured by Cytiva
The ECL plus chemiluminescent system is a laboratory equipment used for the detection and quantification of proteins in Western blot analysis. It generates a luminescent signal in the presence of the target protein, which can be captured and analyzed using specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 mouse mab, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Supersignal west pico chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Bolt 8 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Phosstop, by Roche (1 mentions)
Gar hrp, by Cell Signaling Technology (1 mentions)
Anti mouse caspase 1 p10, by Santa Cruz Biotechnology (1 mentions)
Lc3 antibody, by Santa Cruz Biotechnology (1 mentions)
4 protocols using ecl plus chemiluminescent system
1
Western Blot Analysis of Viral Proteins
2
Western Blot Analysis of EGFR Family
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
15 µg total cell lysates were prepared by briefly sonicating cell pellets in 1× lysis buffer (Cell Signaling) supplemented with a protease inhibitor mixture (Roche) and phosphatase inhibitors (PhosSTOP, Roche). Protein samples were resolved on Bolt 8% Bis–Tris Plus gels (ThermoFisher) and were electrophoretically transferred to a polyvinylidene difluoride membrane and blocked for 1 h at room temperature with 5% dry milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween, pH 7.5). Blots were stained with antibodies against EGFR (D38B1), HER2/ERRB2 (D8F12), HER3/ERRB3(D22C5), and HER4/ERBB4 (111B2),and GAPDH (D16H11) purchased from Cell Signaling Technology and used a dilution of 1:1000 in 5% dry milk in TBST for 1 h at 22 °C. For detection of PHLDA1 expression, blots were stained with 1;1000 dilution of anti-PHLDA1 antibody (EPR6674, abcam®). For detection of PDK4 expression, blots were stained with 2 µg/mL dilution of anti-PDK4 (NBP1-07049, Novus Biologicals). All blots were washed with three changes of TBST for a total of 45 min. Blots were then incubated in TBST buffer with 5% dry milk containing GAR-HRP (1:10,000, Cell Signaling Technology) for 1 h at room temperature. After three washes with TBST, antigen–antibody complexes were detected with the ECL Plus chemiluminescent system (Amersham Biosciences) and visualized with film.
3
Western Blot Analysis of Macrophage Signaling
4
Western Blot Analysis of IL-1RI and Caspase-1 in AM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aliquots of AM lysates were separated on a 10% SDS-PAGE under non-reducing condition. Equivalent loading of the gel was determined by quantitation of protein as well as by re-probing the membranes for actin detection. Separated proteins were electroblotted onto PVDF membrane and blocked for 1 h at room temperature with Tris-buffered saline containing 1% BSA. The membranes were then probed with primary antibody (polyclonal anti-IL-1RI antibody purchased from Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. After washing, primary antibodies associated with the membranes were detected on autoradiographic film by horseradish peroxidase-conjugated secondary antibodies and the ECL plus chemiluminescent system (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions. Blots were quantitated using Scion Image software (Scion Corp., Frederick, MD) and normalized to actin. Caspase-1 cleavage in the AM was measured by detecting its p20 fragment in Western blot using rabbit polyclonal anti-mouse caspase-1 p20 (Santa Cruz Biotechnologies, CA).
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!