Isolated proteins and prestained protein standards (Bio-Rad, Munich, Germany) were subjected to 4.5-15% SDS-polyacrylamide gel electrophoresis under reducing conditions. Proteins were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) for 30 min at 25 V and ≤1 A with the Trans-Blot Turbo Blotting System (Bio-Rad, Munich, Germany). Membranes were blocked for 30 min in blocking buffer (5% dry skim milk+1% BSA in TBS containing 0.1% Tween 20 (TBS-T)), then incubated overnight with primary antibodies (see Table 2 ), washed with TBS-T, and finally incubated at room temperature for 1 h with peroxidase-coupled anti-mouse IgG antibody from donkey (Dianova, Hamburg, Germany, dilution 1 : 1,000) or peroxidase-coupled anti-rabbit IgG antibody from donkey (Amersham Bioscience, Freiburg, Germany, dilution 1 : 10,000). Signals were visualized with Super Signal® West Femto Maximum Sensitive Substrate (Thermo Fisher Scientific, Schwerte, Germany) following the procedure recommended by the supplier and detected with the chemiluminescence detector Fusion-SL4.2 MP (Peqlab Biotechnology, Erlangen, Germany).
Supersignal west femto maximum sensitive substrate
Manufactured by Thermo Fisher Scientific
Sourced in Germany
SuperSignal West Femto Maximum Sensitive Substrate is a chemiluminescent substrate for the detection of low-abundance proteins in Western blotting applications. It provides high sensitivity and a broad linear dynamic range for quantitative analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Prestained protein standards, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Trans blot turbo blotting system, by Bio-Rad (1 mentions)
Originpro 2021 9, by OriginLab (1 mentions)
Pymol 2, by Schrödinger (1 mentions)
Azure 600 western blot imaging system, by Azure Biosystems (1 mentions)
Mouse anti human β actin antibody, by Cell Signaling Technology (1 mentions)
Gradient polyacrylamide gel, by Bio-Rad (1 mentions)
Office scanner, by Epson (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Nupage 4 12 bis tris precast gel, by Thermo Fisher Scientific (1 mentions)
Myogenin, by Abcam (1 mentions)
Desmin, by Abcam (1 mentions)
Las 3000 luminescent image analyzer system, by Fujifilm (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
7 protocols using supersignal west femto maximum sensitive substrate
1
Western Blot Protein Analysis Protocol
2
Synapsin Antibody Binding Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Synapsin slides were blocked for 60 min in 5% (w/v) skimmed milk powder 0.05% Tween20 PBS pH 7.4. Afterward, slides were incubated for 30 min with positive and negative serum as determined with cell-based assays (1:1000 dilution). Washing steps were carried out three times using 0.05% Tween20 PBS. IgG antibodies were detected using goat-anti-human secondary antibodies (Thermo Fisher, #31410, 1:2500). Chemiluminescence signal was detected with an Azure system c400 using SuperSignal West Femto maximum sensitive substrate (Thermo Scientific, Schwerte, Germany). Microarray binding intensities were quantified using the software-tool MARTin (https://github.com/scitequest/martin ) and normalized to the most prominent binder. Heat-maps were generated with OriginPro 2021 9.8.0.200 (OriginLab, Northampton, MA). Human synapsin-I 3D model was extracted from AlphaFold (DeepMind Technologies, London, UK) (Jumper et al., 2021 (link)) database and rendered using Pymol 2.4 (Schrödinger Inc, New York, U.S.).
3
Biotinylated Protein Detection by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 10 μg of protein extracts were separated by 10% SDS-PAGE Tris-glycine polyacrylamide gel electrophoresis, and 0.2 μm nitrocellulose membranes were used for the transfer in Towbin buffer for 4 h at constant 280 mA. Blots were incubated for 5 min with Ponceau S (0.1% (w/v) Ponceau S in 5% glacial acetic acid) for total protein visualization to control for possible loading differences. For immunodetection of biotinylated proteins, membranes were blocked in 7% milk in 1xTBS and 0.01% Tween-20 and streptavidin-HRP immunostaining (1:5,000, Invitrogen cat. #19534–050) was performed at room temperature for 1 h in blocking solution. After 3 washes with TBST, membranes were covered with SuperSignal West Femto Maximum Sensitive Substrate (Thermo Scientific, Cat. #34095) according to manufacturer’s instructions and chemiluminescence was then documented using Azure 600 Western Blot Imaging System (Azure Biosystems).
4
Western Blot Protocol for Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines were lysed using RIPA buffer (Thermo-Fisher Scientific) supplemented with Halt™ protease inhibitor (Thermo-Fisher Scientific). Cell debris was removed after centrifugation at 4°C, and total protein concentration was determined using the DC protein assay (Bio-Rad). Electrophoretic separation of protein (12 μg/well) was performed using 4-15% gradient polyacrylamide gels (Bio-Rad). Separated protein was transferred for 18 hours at 4°C onto PVDF membranes (Bio-rad). Membranes were blocked for one hour at room temperature in TBS containing 0.1% tween (TBS-T) with 5% fat-free milk, followed by overnight incubation at 4°C with mouse anti-human TfR1 antibody (Thermo-Fisher Scientific) (1:500 dilution) or mouse anti-human β-actin antibody (Cell Signaling Technology, Danvers, MA, USA) (1:10,000 dilution) in 5% fat-free milk with TBS-T. Membranes were washed in TBS-T and incubated for 30 minutes at room temperature with a 1:2000 dilution of horseradish peroxidase-conjugated rabbit anti-mouse antibody (Cell Signaling Technology) in 5% milk with TBS-T. Protein signals were developed on X-ray film using the Pierce ECL Western blotting substrate (Thermo-Fisher Scientific) or the SuperSignal West Femto Maximum Sensitive Substrate (Thermo-Fisher Scientific). X-ray film of blots was digitized using an office scanner (Epson, Long Beach, CA, USA).
5
Western Blotting for SMN Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples separated by NuPAGE 4%–12% Bis-Tris precast gels (Life Technologies) were electroblotted onto nitrocellulose membranes. The membrane was probed with monoclonal anti-SMN (BD Transduction Laboratories) and anti-α-tubulin (courtesy of Dr. Muro) as normalization control, followed by horseradish-peroxidase-conjugated rabbit anti-mouse secondary antibody. Protein signals were detected with SuperSignal West Femto Maximum Sensitive Substrate (Thermo Scientific).
6
Analyzing Myogenic Protein Expression via Western Blotting
7
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with lysis buffer II (50mM Tris (pH8), 2% w/v SDS, 5mM EDTA, 3mM EGTA, 25mM NaF, 1mM Na 3 VO 4 ) supplemented with 1mM PMSF, 10 mg/ml aprotinin, and 10 mg/ml leupeptin. Cell homogenates were sonicated for 2 s, clarified by centrifugation and total protein concentration was quantified with DC protein assay. Proteins samples were diluted to the appropriate dilution and mixed with 1/5 volume of 5x SDS-PAGE sample buffer (250mM Tris pH6.8, 10% w/v SDS, 25% v/v glycerol, 500mM DTT, and bromophenol blue). Proteins (30-45 mg per lane) were resolved by SDS-PAGE, electrotransferred to Immobilon-P membrane, and incubated with a primary antibody diluted as recommended by the manufacturer. Membranes were then probed with a horseradish peroxidase-conjugated secondary antibody (Promega V8051, W4011, and W4021; R&D HAF005) and protein signals were developed using the Pierce ECL western blotting substrate (Thermo Scientific; 32106) or the SuperSignal West Femto Maximum Sensitive Substrate (Thermo Scientific; 34095). X-ray films were imaged (digitized) with ChemiDoc System and analyzed with Image Lab Software. Primary antibodies were purchased from Santa Cruz biotechnology (p21, sc-6246; p53, sc-6243; GAPDH, sc-25778), Cell signaling (phospho-S6 Ser235/236, 2211 , S6, 2317; phospho-p65, 3033; p65, 8242) , and Sigma Aldrich (b-actin, A5441).
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!