The cells were lysed using 1× cell lysis buffer (Cell Signaling Technology, Inc.), including 1 mM phenylmethylsulfonyl fluoride, and transparent protein lysates were obtained following centrifugation at 29,994 × g. Bradford assay reagent (Fisher Scientific International Inc.) was used for protein quantitation, and 40-μg protein samples were separated using 5% stacking and 10% resolving gels at 60 V for 1 or 2 h. The separated proteins were immunoblotted to a polyvinylidene difluoride membrane using iBlot® dry blotting system (Invitrogen Life Technologies) and blocked for 1 h with 5 % skimmed milk in Tris-buffered saline and 1% Tween 20 (Sigma-Aldrich). Protein bands were visualized with the SuperSignal West Femto Chemiluminescence kit (Fisher Scientific International Inc.) and analyzed using Kodak Gel Logic 1500 imaging system software (Eastman Kodak Co., Rochester, NY, USA). For all experiments, a minimum of three independent experiments were performed and representative results were selected for analysis.
Supersignal west femto chemiluminescence kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperSignal West Femto chemiluminescence kit is a laboratory product designed for the detection of low-abundance proteins in Western blot analyses. It provides a sensitive and efficient method for visualizing and quantifying target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (9 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (5 mentions)
Mouse anti actin, by Santa Cruz Biotechnology (4 mentions)
Rabbit anti scf, by Abcam (4 mentions)
A18 4, by Thermo Fisher Scientific (4 mentions)
Lds loading buffer, by Thermo Fisher Scientific (3 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Imagequant tl software, by GE Healthcare (3 mentions)
Las 4000, by Cytiva (3 mentions)
Bn1002box, by Thermo Fisher Scientific (2 mentions)
Np0336box, by Merck Group (2 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (2 mentions)
0.2 μm pvdf membrane, by Bio-Rad (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (2 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (2 mentions)
H3k4me2 c75h12, by Cell Signaling Technology (2 mentions)
H3k36me2 c64g9, by Cell Signaling Technology (2 mentions)
Ac 74, by Merck Group (2 mentions)
Nupage bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Supersignal western blot enhancer kit, by Thermo Fisher Scientific (2 mentions)
25 protocols using supersignal west femto chemiluminescence kit
1
Western Blot Protein Analysis Protocol
2
Immunoblotting of HLMVEC Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates obtained from HLMVEC or lung tissues were subjected to SDS-PAGE in 4–20% Tris-glycine gels (Cat # 4561094) (Bio-Rad, Hercules, CA) followed by electrotransfer to PVDF membranes (Cat # 1704156) (Bio-Rad). Specific protein bands were detected using respective primary and secondary antibodies and visualized using Super Signal West Femto Chemiluminescence kit (Cat # PI34095) (Fisher Scientific, Hampton, NH) using LI-COR Odyssey image station (Lincoln, NE). Specific protein images obtained were quantified using LI-COR Image Station software. Protein expressions were normalized by re-probing membranes with an anti-β-actin antibody (Sigma-Aldrich).
3
Bone Marrow Protein Extraction and Western Blot
4
Western Blot for Hematopoietic Stem Cells
5
Splenic Endothelial Cell Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 30,000 CD45−Ter119−VE-cadherin+ splenic endothelial cells were flow cytometrically sorted into 50 μl of 66% Trichoracetic acid (TCA) in water. Extracts were incubated on ice for at least 15 min and centrifuged at 16,100 × g at 4°C for 10 min. Precipitates were washed in acetone twice and the dried pellets were solubilized in 9M urea, 2% TritonX-100, and 1% DTT. Samples were separated on 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. Blots were developed with the SuperSignal West Femtochemiluminescence kit (Thermo Scientific). Primary antibodies used: rabbit-anti-SCF (Abcam, 1:1000) and mouse-anti-Actin (Santa Cruz, clone AC-15, 1:20,000).
6
Calu-3 Cell Protein Expression Analysis
7
TCA Precipitation and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as described previously (Xu et al., 2020 (link)). Approximately 5 × 105 DN3 cells were sorted directly into 250 μl PBS containing 20% trichoracetic acid (TCA). The concentration of TCA was adjusted to 10% after sorting and cells were incubated on ice for 30 min before centrifugation at 13,000 rpm for 10 min at 4°C. Precipitates were washed twice with pure acetone (Fisher scientific, A18-4) and solubilized in 9 M urea, 2% Triton X-100, and 1% dithiothreitol (DTT) in 1 x LDS sample buffer (Invitorgen, NP0007). Samples were separated on NuPAGE 4–12% Bis-Tris protein gels (Invitrogen, NP0336BOX) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. Blots were developed with the SuperSignal West Femto chemiluminescence kit (Thermo Scientific, 34096). Antibodies and reagents used are in Appendix 1. The original western blot images are in source data files.
8
Bone Marrow Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow was flushed out of the bone and then dissociated in 66% Trichoracetic acid (TCA) in water. Extracts were incubated on ice for at least 15 min and centrifuged at 16,100 × g at 4°C for 10 min. Precipitates were washed in acetone twice and the dried pellets were solubilized in 9M urea, 2% TritonX-100, and 1% DTT. Samples were separated on 4–12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. Blots were developed with the SuperSignal West Femtochemiluminescence kit (Thermo Scientific). Primary antibodies used: rabbit-anti-SCF (Abcam, catalogue number ab64677, 1:1000) and mouse-anti-Actin (Santa Cruz, clone AC-15, 1:20,000).
9
Splenic Endothelial Cell Protein Extraction and Analysis
10
Western Blot Analysis of NF-κB and ERK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates from treated PSCs were run on a 4 ~ 12% Bis-Tris gel then transferred to a 0.2 mm nitrocellulose membrane. After blocking in 5% milk, membranes were incubated overnight at 4°C with primary antibodies for phospho-NF-κB p65 (93H1), NF-κB p65 (L8F6), phosphor-ERKd13.14.4E), ERK (137F5) (All from Cell Signaling Technology) and B-actin (Sigma, A5316). Membranes were washed then incubated with peroxidase-conjugated goat-anti-rabbit-HRP and goat-anti-mouse-HRP secondary antibodies (Fisher Thermo, 31430) for 1 h at room temperature then developed with SuperSignal® West Femto chemiluminescence kit (Thermo Scientific, 34095) and exposed on film.
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!