Supersignal west femto enhanced chemiluminescent substrate

Manufactured by Thermo Fisher Scientific

SuperSignal West Femto enhanced chemiluminescent substrate is a laboratory reagent used for the detection of proteins in Western blot analysis. It generates a sensitive chemiluminescent signal that can be detected using a compatible imaging system.

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Lab products found in correlation

Disuccinimidyl suberate, by Merck Group (1 mentions) Anti ha, by Thermo Fisher Scientific (1 mentions) N glycosidase f, by New England Biolabs (1 mentions) Ibright cl1000 imaging system, by Thermo Fisher Scientific (1 mentions) Anti rabbit or anti mouse secondary antibody, by Merck Group (1 mentions) Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions) Tris hcl gel, by Bio-Rad (1 mentions) Complete mini edta free protease inhibitor tablet, by Roche (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions)

3 protocols using supersignal west femto enhanced chemiluminescent substrate

Western Blot Analysis of Protein Tags

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Medium and cell lysates were subjected to SDS-PAGE. Membranes were blocked with fat-free milk (5%) with Tween (1%) for 1 hour and incubated overnight at 4°C in the presence of either a monoclonal mouse anti-V5 (Sigma-Aldrich) or anti-HA (Invitrogen) antibody. Blots were visualized using a goat antimouse horseradish peroxidase-conjugated antibody and SuperSignal West Femto enhanced chemiluminescent substrate (Thermo Scientific) according to manufacturer's instructions. Conditioned media were deglycosylated using N-glycosidase F (New England Biolabs) or covalently cross-linked with disuccinimidyl suberate (Sigma-Aldrich), according to manufacturer's instructions, and the products analyzed by Western blotting.

Bassett J.H., van der Spek A., Logan J.G., Gogakos A., Bagchi-Chakraborty J., Murphy E., van Zeijl C., Down J., Croucher P.I., Boyde A., Boelen A, & Williams G.R. (2015). Thyrostimulin Regulates Osteoblastic Bone Formation During Early Skeletal Development. Endocrinology, 156(9), 3098-3113.

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Arabidopsis CCV Purification and Analysis

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CCVs were purified from suspension-cultured Arabidopsis T87W cells, as previously described (29 (link)). Equal amounts of protein from the deuterium ficoll gradient load (DFGL) and purified CCV samples were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, and immunoblotted with anti-CLC2 1:10,000 (30 (link)), anti-CHC 1:1,000 (sc-57684, Santa Cruz Biotechnology), anti-AP2mu2 1:250 (31 (link)), anti-TPLATE 1:2,000 (32 (link)), and anti-DRP1c 1:500 (33 (link)) antibodies. Primary antibodies were detected through anti-rabbit or anti-mouse secondary antibodies (Sigma-Aldrich) conjugated to horseradish peroxidase at 1:5,000 before application of SuperSignal West Femto enhanced chemiluminescent substrate (Thermo Fisher) and subsequent imaging with iBright CL1000 Imaging System (Thermo Fisher Scientific). The integrated density values of the chemiluminescent bands of the DFGL and CCV fractions were measured by ImageJ (NIH). The integrated density value of the CCV band was divided by the corresponding value of the DFGL band to determine the relative enrichment across three independent CCV purifications.

Johnson A., Dahhan D.A., Gnyliukh N., Kaufmann W.A., Zheden V., Costanzo T., Mahou P., Hrtyan M., Wang J., Aguilera-Servin J., Van Damme D., Beaurepaire E., Loose M., Bednarek S.Y, & Friml J. (2021). The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis. Proceedings of the National Academy of Sciences of the United States of America, 118(51), e2113046118.

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Neutrophil Protein Quantification and Western Blot

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Neutrophil lysates were prepared by lysing cells in lysis buffer (50 mmol·L⁻¹ Tris pH 7.4, 150 mmol·L⁻¹ NaCl, 0.5% NP-40, 5 mmol·L⁻¹ MgCl₂) containing complete mini EdTA-free protease inhibitor tablet (Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (final concentration 100 μm Na₄P₂O₇, 1mm NaF, 1 mm Na₃VO₄, 300 μm β-glycerophosphate, 2 mm imidazole, 4 mm sodium tartrate). Protein quantification was performed using a bicinchoninic acid assay kit (Thermo Fisher), and equivalent amounts of total cell protein were separated by SDS/PAGE on precast 7.5% Tris–HCl gels (BioRad, Hercules, CA, USA). Following transfer to 0.45 μm nitrocellulose, blots were blocked in 5% nonfat dry milk (NFDM) in 1× TBS plus 0.1% Tween 20 (TBST). Primary rabbit anti-KRIT1 antibody (Abcam) was diluted in TBST plus 5% NFDM and the membrane was incubated in primary antibody overnight at 4 °C. The membrane was then incubated with anti-rabbit-HRP secondary antibody for 1 h at room temperature before detection using SuperSignal West Femto enhanced chemiluminescent substrate (ThermoFisher Scientific). Densitometry was performed using a BioRad ChemiDoc Imaging System to quantify band intensity.

Nobiletti N., Liu J, & Glading A.J. (2022). KRIT1-mediated regulation of neutrophil adhesion and motility. The FEBS journal, 290(4), 1078-1095.

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