Cells were lysed in IP Lysis/Wash buffer (Thermo Fisher Scientific) supplemented with 5% (vol/vol) Protease Mixture Inhibitor (MilliporeSigma) and 1 mM phenylmethanesulfonyl (MilliporeSigma). After two homogenization cycles (7 s) with an ultrasonic cell disruptor (Microson; Misonix), total cell lysates were centrifuged at 18,000g for 10 minutes. The supernatant containing proteins was collected and the protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Immunoblotting analysis was carried out as previously described (8 (link)). Antibodies against Actin (MilliporeSigma, A2066; dilution 1:4000), DOT1L (Cell Signaling, 77087; dilution 1:1000), HIF1A (Abcam, ab82832; dilution 1:1000), total H3 (Abcam, ab1791; dilution 1:10,000), and H3K79me2 (Abcam, ab3594; dilution 1:1000) were used following the manufacturer’s instructions. The blotting signals were detected using the SuperSignalWest Femto Maximum Sensitivity Substrate system (Thermo Fisher Scientific).
Supersignalwest femto maximum sensitivity substrate system
Manufactured by Thermo Fisher Scientific
The SuperSignalWest Femto Maximum Sensitivity Substrate system is a chemiluminescent substrate designed for the detection of proteins in Western blot applications. It provides high sensitivity for the detection of low-abundance proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ab1791, by Abcam (2 mentions)
Ip lysis wash buffer, by Thermo Fisher Scientific (2 mentions)
Microson, by Bioventus (2 mentions)
Phenylmethanesulfonyl, by Merck Group (2 mentions)
A2066, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab3594, by Abcam (1 mentions)
Ab82832, by Abcam (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Ab3594, by Cell Signaling Technology (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Mcl 1 clone s 19, by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Pierce ecl western blotting substrate system, by Thermo Fisher Scientific (1 mentions)
M per buffer, by Thermo Fisher Scientific (1 mentions)
Na3vo4, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
5 protocols using supersignalwest femto maximum sensitivity substrate system
1
Protein extraction and immunoblotting
2
Immunoblotting Analysis of Apoptotic Proteins
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Differentiated THP-1 cells were lysed with a cell lysis buffer, and protein concentration of cell lysates was determined using the Bradford protein assay (Bio-Rad) (Chan et al., 1997 (link)). Thirty micrograms of protein for each condition was resolved by 12% SDS–PAGE and transferred onto iBLOT PVDF membrane using transfer machine from Invitrogen (Thermo Fisher Scientific). After the membrane were blocked in 5% of dry milk for 1 h, they were probed with cytochrome C, Bax, Bak, or β-actin antibodies at a dilution of 1:1000 (v/v), followed by detection with HRP-conjugated anti-rabbit IgG (1:2000 dilution). The bands were visualized by using of SuperSignal West Femto Maximum Sensitivity Substrate System (Thermo Fisher Scientific).
3
Quantitative Immunoblotting Analysis
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Cells were homogenized in IP Lysis/Wash buffer (Thermo Fisher) supplemented with 5% (vol/vol) Protease Mixture Inhibitor (Sigma) and 1 mM phenylmethanesulfonyl (Sigma). After two homogenization cycles (7 s) with an ultrasonic cell disruptor (Microson; Misonix), total cell lysates were centrifuged 10 min at 18,000g, and supernatant containing proteins was collected. The protein concentration of the extracts was determined by Pierce BCA Protein Assay Kit (Thermo Scientific). Immunoblotting analyses were performed as described in previous studies3 (link). Antibodies against Actin (Sigma, A2066; dilution 1:4,000), active b-catenin (Merck Millipore, 05-665, clone 8E7; dilution 1:1,000), total β-catenin (BD biosciences, 610154, clone 14/β-catenin; dilution 1:2,000), DOT1L (Bethyl, A300-954A; dilution 1:1,000), GAPDH (Ambion, AM4300, clone 6C5; dilution 1:10,000), total H3 (Abcam, ab1791; dilution 1:10,000), H3K79me2 (Abcam, ab3594; dilution 1:1,000), and SIRT1 (Cell signalling, 2496, clone C14H4; dilution 1:1,000) were used following manufacturer’s instructions. The blotting signals were detected using the SuperSignalWest Femto Maximum Sensitivity Substrate system (Thermo Scientific).
4
Western Blot Analysis of Protein Markers
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Cells were lysed in M-PER buffer (Pierce Biotechnology, Rockford, IL, USA) supplemented with protease and phosphatase inhibitor cocktails (Sigma). Total cell extracts were separated using SDS-PAGE in 10% gels, transferred to nitrocellulose membranes, and blotted as described previously [97 (link)]. The primary antibodies included anti-NGAL (clone AF1757, goat IgG, specific for the dimeric and monomeric forms; R&D Systems), anti-MMP-9 (clone EP1254, rabbit Ig, specific for CPX; Abcam, Paris, France), anti-NGAL-R/Slc22A17 (clone 75010, rabbit Ig; Biorbyt), and antibodies against Mcl-1 (clone S-19, rabbit IgG; Santa-Cruz), Bcl-2 (clone 100, mouse IgG1; Santa-Cruz), STAT3 (clone C20, rabbit IgG; Santa-Cruz), phospho-Tyr705-STAT3 (clone 710093, rabbit Ig; Thermo Fisher Scientific, Waltham, MA, USA), STAT1 (clone -23, rabbit IgG; Santa-Cruz) and actin (clone C4, mouse IgG1; ICN Biomedicals, Solon, OH, USA). Immunoreactive proteins were detected using horseradish peroxidase-conjugated secondary antibodies and visualized with the Pierce ECL Western blotting substrate system or the SuperSignal West Femto Maximum Sensitivity Substrate system (both from Thermo Fisher Scientific). Immunoblot images were acquired in an MF-ChemiBIS 4.2 imager (DNR Bio-Imaging Systems Ltd., Neve Yamin, Israel) and quantified using ImageJ64 software.
5
Protein Extraction and Western Blot Analysis
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Cells were harvested and resuspended in RIPA buffer (Thermo Scientific) supplemented with 1 mM phenylmethanesulfonyl (Sigma), 5% protease inhibitor cocktail (Sigma), 2.3 mM Na 3 VO 4 (Sigma) and 5 mM NaF (Merck Millipore). Cell lysates were sonicated (two cycles of 7 s) and centrifuged at 18.000 g for 10 min, and supernatant containing proteins was collected. Protein concentration of the extracts was determined by Pierce BCA Protein Assay Kit (Thermo Scientific). Immunoblotting analyses were performed as previously described 19 . Antibodies against Actin (Sigma, A2066; dilution 1:4.000), active b-catenin (Merck Millipore, 05e665, clone 8E7; dilution 1:1.000) and total b-catenin (BD biosciences, 610154, clone 14; dilution 1:2.000) were used. Blotting signals were detected using the SuperSignalWest Femto Maximum Sensitivity Substrate system (Thermo Scientific).
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