Supersignal west femto chemiluminescence substrate

Manufactured by Thermo Fisher Scientific

Sourced in United States

SuperSignal West Femto Chemiluminescence Substrate is a sensitive luminol-based chemiluminescent substrate used for the detection of proteins in Western blotting. It is designed to provide a strong, long-lasting signal that can be detected using a luminometer or X-ray film.

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Lab products found in correlation

Protease inhibitor cocktail, by Roche (3 mentions) Hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions) West clarity max substrate, by Bio-Rad (2 mentions) Phosphatase inhibitor cocktail, by Roche (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Alexa 488 hydrazide, by Thermo Fisher Scientific (1 mentions) Typhoon trio imager, by GE Healthcare (1 mentions) Gel doc xr system, by Bio-Rad (1 mentions) Anti h3k27me3 antibody, by Diagenode (1 mentions) Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions) Cox 4, by Abcam (1 mentions) Supersignal west pico, by Thermo Fisher Scientific (1 mentions) Eif2α, by Abcam (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Vertical electrophoresis cell, by Bio-Rad (1 mentions) Immobilon p membrane, by Merck Group (1 mentions) Ab1791 100, by Abcam (1 mentions) C15410195, by Diagenode (1 mentions)

13 protocols using supersignal west femto chemiluminescence substrate

Immunoblotting with Phosphatase and Protease Inhibitors

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Cells were lysed on ice in RIPA buffer (10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholic acid, 0.1% SDS, protease inhibitor cocktail (1xPIC, Merck, P8340)), containing 10 mM sodium butyrate. After sonication and centrifugation, the protein contents of the supernatants were determined prior to SDS-PAGE and immunoblotting. To verify equal loading the membranes were reversibly stained with 0.2% Ponceau S. The antibodies were applied in 5% low-fat milk, 0.05% Tween20 in PBS. For analyzing p38 phosphorylation, the lysis buffer contained additionally a phosphatase inhibitor cocktail (1 mM ortho-vanadate, 50 nM okadaic acid, 50 nM β-phospho-glycerol, 25 mM NaF, 5 mM EGTA). The antibodies were applied in 5% BSA, 0.05% Tween20 in PBS. The blots were developed with the SuperSignal West Femto Chemiluminescence Substrate (ThermoFisher Scientific, 34094). For details of the antibodies see Table 2.
Monoclonal antibodies recognizing ASH2L were generated as described before (20 (link)). Clone 5E10 recognizes an epitope in the N-terminal fragment of ASH2L, which is conserved in the mouse.

Bochyńska A., Stenzel A.T., Sayadi Boroujeni R., Kuo C.C., Barsoum M., Liang W., Bussmann P., Costa I.G., Lüscher-Firzlaff J, & Lüscher B. (2022). Induction of senescence upon loss of the Ash2l core subunit of H3K4 methyltransferase complexes. Nucleic Acids Research, 50(14), 7889-7905.

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Quantitative Alexa488 Labeling and Western Blotting

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Whole brain homogenates (2 mg/mL) were incubated with 500 μM Alexa488 hydrazide (ThermoFisher) in 0.1 M phosphate buffer (pH = 5.5) for 2 h at room temperature. These proteins were then separated on a 4-12% Bis-Tris, SDS-PAGE gel (Invitrogten) with 30 μg of protein. Gels were immediately imaged using a GE Typhoon Trio imager. Western Blots: Lysates were stored at −20 °C before use. Equal amounts of protein were incubated under reducing conditions (1% β-mercaptoethanol) with 4X NuPAGE LDH sample buffer (ThermoFisher), and run on 4-12% SDS NuPage Gels (Invitrogen). Proteins were transferred to PVDF membranes using the iBLOT2, blocked in 5% non-fat milk for 1 h in room temperature, and probed with the appropriate primary antibodies overnight at 4 °C (1:500–1:2000 in blocking buffer). The following day, membranes were washed 3x with TBS-T (TBS + 0.1% Tween-20) for 5 min each and probed with an HRP-linked secondary antibody (1:1000 in blocking buffer) for 1 h in room temperature, then washed again 3x with TBS-T. Gels were visualized using SuperSignal West Femto Chemiluminescence substrate (ThermoFisher) on the Gel Doc XR + System (Biorad). Membranes were re-probed for β-actin (loading control) and stored at 4 °C until imaging. All values were normalized to expression levels of β-actin and normalized to control at 1.

Knopp R.C., Lee S.H., Hollas M., Nepomuceno E., Gonzalez D., Tam K., Aamir D., Wang Y., Pierce E., BenAissa M, & Thatcher G.R. (2020). Interaction of oxidative stress and neurotrauma in ALDH2−/− mice causes significant and persistent behavioral and pro-inflammatory effects in a tractable model of mild traumatic brain injury. Redox Biology, 32, 101486.

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Quantifying H3K27me3 Protein Levels

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Pre-incubation of anti-H3K27me3 antibody (C15410195, Diagenode) and H3K27me3 peptide (Intavis) was done for 2 h in RT in PBST, with occasional mixing. The peptide-to-antibody molar ratio was 50:1. A control solution – PBST with just antibody at 2X final concentration – was used at the same time. At the end of the pre-incubation, 4% BSA blocking solution was added to the peptide/antibody mixture to a final concentration of 3% BSA, mixed briefly and added to the membranes. After washing three times for 10 min in PBST, membranes were incubated with peroxidase-labeled secondary antibody for 1 h at room temperature. The membranes were washed three times for 10 min in PBST, incubated with SuperSignal West Femto Chemiluminescence Substrate (Thermo Scientific) for 1 min, and exposed for 1–2 min.

Mikulski P., Komarynets O., Fachinelli F., Weber A.P, & Schubert D. (2017). Characterization of the Polycomb-Group Mark H3K27me3 in Unicellular Algae. Frontiers in Plant Science, 8, 607.

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Protein Extraction and Western Blot Analysis

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Lung tissue was homogenized in Cytokine buffer containing NaCl, Tris and Tween-20 supplemented with Protease Inhibitor Cocktail (Cat # 04693159001, Roche) and Phosphatase Inhibitor Cocktail (Cat # 04906837001, Roche). Cell lysates were prepared by harvesting cells in Cell Lysis Buffer (Cat. # 9803, Cell Signaling Technologies) supplemented with Protease and Phosphatase Inhibitor. The lysates were cleared of debris by centrifugation and total protein estimation was performed on the collected supernatant by Bicinchoninic Acid (BCA) assay. Proteins were resolved by loading 15-20 μg of protein on a 10%SDS-PAGE Gel. The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane by using a wet transfer technique. Blots were probed with antibodies against the protein of interest overnight at 4°C, followed by HRP-conjugated Secondary Antibody (Cat # 111035003, Jackson ImmunoResearch Laboratories) for 1hr at RT. The blots were developed using Super Signal West Femto-chemiluminescence substrate (Cat # 34095, ThermoFisher Scientific) or Bio-Rad West Clarity Max substrate (Cat # 1705062) Quantification was done using ImageJ.

Garg M., Johri S., Sagar S., Mundhada A., Agrawal A., Ray P, & Chakraborty K. (2021). Cardiolipin-mediated PPARγ S112 phosphorylation impairs IL-10 production and inflammation resolution during bacterial pneumonia. Cell reports, 34(6), 108736.

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Western Blot Analysis of Sonoporation

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Samples for western blot were harvested 2 h post sonoporation, washed twice in PBS and lysed in RIPA buffer containing protease- and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was quantified in accordance with Bio-Rad DC protein assay instruction manual for microtiter plates. For the western blots 10% SDS-polyacrylamide gels were loaded with 20–30 μg protein per well. Proteins were separated at 100–120 V and transferred to a nitrocellulose membrane (240 mA, 150 min, 4 °C), followed by 1 h blocking using 5% skim milk powder in Tris-buffered saline (TBS) + 1% Tween 20. Primary antibodies were diluted in 5% skim milk in TBS-Tween and incubated over night at 4 °C. Primary antibodies used were p-eIF2α Ser-51 (Abcam, Cambridge, UK) and eIF2α (Abcam). COX IV (Abcam) was used as loading control. Secondary antibodies were diluted in 5% skim milk powder in TBS-Tween 20 and incubated for 1 h at room temperature. Membranes were developed using SuperSignal^® West Pico or SuperSignal^® West Femto Chemiluminescence Substrate (Thermo Fischer Scientific) in accordance with manufacturers recommendations.

Haugse R., Langer A., Gullaksen S.E., Sundøy S.M., Gjertsen B.T., Kotopoulis S, & McCormack E. (2019). Intracellular Signaling in Key Pathways Is Induced by Treatment with Ultrasound and Microbubbles in a Leukemia Cell Line, but Not in Healthy Peripheral Blood Mononuclear Cells. Pharmaceutics, 11(7), 319.

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Western Blot Analysis of H3K27me3

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Around 50.000 algae cells were diluted in Laemmli’s sample buffer, and 3–7 μl of total protein/lane was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% SDS-polyacrylamide gel. The resolved proteins were transferred onto polyvinylidenedifluoride (PVDF) membranes (Immobilon-P membrane, Milipore) using a Vertical Electrophoresis Cell (Bio-Rad). Following transfer, membranes were washed with phosphate buffered saline containing 0.05% Tween-20 (PBST) and then blocked in 5% milk diluted in PBST for 1 h at room temperature. The membranes were then incubated overnight with anti_H3K27me3 (N 07-449, Millipore or C15410195, Diagenode) and anti H3 (ab1791-100, Abcam) rabbit polyclonal antibodies 1:2000 in PBST containing 5% milk at 4°C. After washing three times for 10 min in PBST, membranes were incubated with peroxidase-labeled secondary antibody for 1 h at room temperature. The membranes were washed three times for 10 min in PBST, incubated with SuperSignal West Femto Chemiluminescence Substrate (Thermo Scientific) for 1 min, and exposed for 1–2 min. Commercial histone extract from calf thymus (Sigma, #9064-47-5) was used as a positive control for H3K27me3 detection.

Mikulski P., Komarynets O., Fachinelli F., Weber A.P, & Schubert D. (2017). Characterization of the Polycomb-Group Mark H3K27me3 in Unicellular Algae. Frontiers in Plant Science, 8, 607.

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Biotinylation of K5 Polysaccharide

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K5 polysaccharide (4 mM) was biotinylated at the reducing end through a reaction with 1:1 molar ratio of N-(aminooxyacetyl)-N’-(D-Biotinoyl) hydrazine in the presence of 100 mM aniline, in 100 mM NaOAc pH 4.6^{59 (link),60 (link)}. The reaction was incubated for 48 h at 37 °C before free biotin was removed by desalting on a C 10/40 column packed with Sephadex G25 (Cytiva), pre-equilibrated in dH₂O. Separation was conducted over 1.2 CV with inline UV monitoring at 232 and 210 nm. Fractions eluting in the void volume corresponding to K5 were pooled and lyophilized. Biotinylation was assessed by dot blot; 2 µL of 1 mg mL⁻¹ K5 or biotinylated K5 was spotted onto a nitrocellulose membrane and allowed to dry, before being blocked with 5% (w/v) BSA in PBS-T (PBS, 0.05% Tween-20) for 1 h. The blocked nitrocellulose membrane was washed 3x with PBS-T before being incubated with 1:2000 HRP-streptavidin (Thermo 21124) in 1% (w/v) BSA in PBS-T for 30 min. Following three washes with PBS-T, the presence of biotin was detected by the addition of SuperSignal West Femto chemiluminescence substrate (Thermo), and imaged using a ChemiDoc system (BioRad).

Mycroft-West C.J., Abdelkarim S., Duyvesteyn H.M., Gandhi N.S., Skidmore M.A., Owens R.J, & Wu L. (2024). Structural and mechanistic characterization of bifunctional heparan sulfate N-deacetylase-N-sulfotransferase 1. Nature Communications, 15, 1326.

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Notch1 Signaling Western Blot

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Whole-cell lysates were prepared with RIPA buffer with protease inhibitors. Protein concentration was determined with the Bio-Rad protein assay dye reagent (Bio-Rad). Proteins were separated using SDS-PAGE and transferred to PVDF membranes. Antibodies used for Western blot were cleaved Notch1 (Val1744) antibody (Cell Signaling Technology catalog 2421), GAPDH (MilliporeSigma catalog AB2302), and secondary anti-mouse–HRP (Bio-Rad catalog 170-6516) or anti-rabbit–HRP (Bio-Rad catalog 170-6515). Blots were visualized with SuperSignal west pico chemiluminescence (Thermo Fisher Scientific) or SuperSignal west femto chemiluminescence substrate (Thermo Fisher Scientific).

Sottoriva K., Paik N.Y., White Z., Bandara T., Shao L., Sano T, & Pajcini K.V. (2022). A Notch/IL-21 signaling axis primes bone marrow T cell progenitor expansion. JCI Insight, 7(9), e157015.

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Immunoblot and In Vitro Kinase Assay

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The c-fos-luc promoter (pFos WT-GL3) was provided by Dr. Ron Prywes (Columbia University, New York, NY). The AP-1 luciferase reporter plasmid (pGL4.44[luc2P/AP1 RE/Hygro] vector; Cat. No., E4111) was purchased from Promega (Madison, WI, USA).
Immunoblot assay. The cells were disrupted in RIPA buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.25% sodium deoxycholate, 1 mM EDTA, 1% NP40, 1 mM NaF, 0.2 mM phenylmethyl sulfonyl fluoride, 0.1 mM sodium orthovanadate, and a protease inhibitor cocktail (Roche Life Science, Indianapolis, IN, USA). The proteins were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA) blocked in 5% skim milk and probed with the indicated antibodies. The immunoblots were visualised using a SuperSignal West Femto chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA) and detected using the LAS 4000-mini biomolecular imaging system (FUJIFILM, Tokyo, Japan).
In vitro kinase assay. Kinase assay was performed as described previously (24) (link). Briefly, the Reaction Biology Corp. Kinase HotSpotSM service was used for the kinase assay using 1 μM concentration of ATP.

Bhattarai P.Y., Oh C.H., Kim G., Kim M.S., Lee B.S, & Choi H.S. (2020). Novel Imidazo[2,1-b]oxazole Derivatives Inhibit Epithelial Cell Transformation and Triple Negative Breast Cancer Tumorigenesis. Anticancer research, 40(9).

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Immunoblotting Analysis of Schistosoma Proteins

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To analyse the proteins by immunoblotting they were first resolved by 12% SDS-PAGE. Proteins were transferred to nitrocellulose using a semi-dry blotting apparatus. The nitrocellulose membrane was blocked for 1hr at RT with 15 ml of 5% milk in TBS/0.05% Tween-20. Then, 15ml of 2.5% milk in TBS/0.05% Tween-20 containing the primary antibody (anti-Sm16 or serum from infected mice) was added to the nitrocellulose membrane for 1 hr, with rotation at RT. The nitrocellulose was washed three times for five min each with TBS/0.05% Tween-20 and then incubated in 15 ml of secondary antibody-peroxidase conjugate in TBS/0.05%-Tween for 1hr at RT. The nitrocellulose was washed three times for five min each with TBS/0.05 Tween-20 and then incubated in 15 ml of secondary antibody-peroxidase conjugate in TBS/0.05%-Tween for 1 hr at RT. The nitrocellulose filter was again washed three times for five min each. Bound antibody was visualized by adding 1 ml of each reagent of SuperSignal West Femto Chemiluminescence Substrate (ThermoFisher Scientific, USA) for 5 minutes. The membrane was dried and developed in the dark using the autoradiography cassette and Kodak X-OMAT 2000 processor system.

Shiels J., Cwiklinski K., Alvarado R., Thivierge K., Cotton S., Gonzales Santana B., To J., Donnelly S., Taggart C.C., Weldon S, & Dalton J.P. (2020). Schistosoma mansoni immunomodulatory molecule Sm16/SPO-1/SmSLP is a member of the trematode-specific helminth defence molecules (HDMs). PLoS Neglected Tropical Diseases, 14(7), e0008470.

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