Proteome profiler human protease array kit and proteome profiler human protease inhibitor array kit (R&D Systems) were used to detect 34 proteases and 32 inhibitors, respectively. HFDa pooled fractions were dried by vacuum concentration (miVac speed trap, GeneVac Ltd, Ipswich, UK) and resuspended in 50 μL of 0.3% (w/v) sodium dodecyl sulphate (SDS) for 2 hours. Samples were diluted to 1 mL with Array buffer 6 plus 0.5 mL of Array buffer 4 (both buffers from R&D Systems). Fifteen microliters of reconstituted detection antibody cocktail was added to the sample solution and incubated for 1 hour at RT in end-over-end agitation. Mix sample-antibody solutions were incubated overnight at 6°C with the membrane on a rocking platform shaker. After three 10-minute washes with 1x buffer, the membranes were incubated with infrared dye-coupled streptavidin with a fluorescent tag emitting at 800 nm (LI-COR Biosciences), 1 : 2000 dilution for 1 h at room temperature on the rocking platform shaker. After three × 10 min wash with 1x Wash Buffer the membrane slides were acquired by Odyssey Infrared Laser Scanner (LI-COR Biosciences) and relative quantification analysis was performed by Odyssey Infrared Laser Scanner software (LI-COR Biosciences).
Odyssey infrared laser scanner
Manufactured by LI COR
Sourced in United States
The Odyssey Infrared Laser Scanner is a laboratory equipment designed for fluorescence-based detection and quantification of proteins, nucleic acids, and other biomolecules. It utilizes infrared laser technology to excite fluorescent dyes and capture high-resolution digital images of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Synergy h4 hybrid plate reader, by Agilent Technologies (2 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine lipids, by Avanti Polar Lipids (2 mentions)
Array buffer 6, by R&D Systems (1 mentions)
Proteome profiler human protease array kit, by R&D Systems (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
0.22 μm pvdf membrane, by Merck Group (1 mentions)
Ab237703, by Abcam (1 mentions)
Ab138515, by Abcam (1 mentions)
Ab0035, by Abways (1 mentions)
Cy5040, by Abways (1 mentions)
Ab0054, by Abways (1 mentions)
Goat anti rabbit igg dylight 680, by Rockland Immunochemicals (1 mentions)
Goat anti mouse igg dylight 800, by Rockland Immunochemicals (1 mentions)
6 protocols using odyssey infrared laser scanner
1
Protease and Inhibitor Profiling
2
Reconstitution and Kinetics of Rhomboid Proteases
3
NF-κB Signaling Pathway Protein Analysis
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RIPA buffer (Solarbio, Beijing, China) was utilized to separate total proteins from cell lysates. After resolution using 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, proteins were shifted onto 0.22 μm PVDF membranes (Millipore). Following 1 h of blocking in 1× TBST buffer containing 5% nonfat dry milk, an overnight incubation of the membranes was performed at 4°C using primary antibodies to NF-κB p65 (#8242, 1:1000, CST, USA), IκBα (#4814, 1:1000, CST, USA), p-IκBα (#2859 1:1000, CST, USA), IKKβ (#8943 1:1000, CST, USA), p-IKKα/β (#2697 1:1000, CST, USA), NF-κB p50 (CY5040, 1:2000, Abways, China), RAB10 (ab237703, 1:1000, Abcam, USA), E2F2 (ab138515, 1:1000, Abcam, USA), Lamin B1 (AB0054, 1:1000, Abways, China) and β-actin (AB0035, 1:1000, Abways, China). After a triple ten-minute rinse using 1× TBST buffer, the membranes were proceeded 1 hour incubation at 37°C protected from light utilizing the corresponding DyLight 800-labeled goat anti-mouse or anti-rabbit antibodies (1:7000, abbkine, China). Finally, the blot image was recorded with the Odyssey Infrared Laser Scanner (LICOR Biosciences). Three replicates were required for each experiment. β-Actin levels were employed to normalize protein levels of the whole cell lysis and Lamin B1 for nuclear extract.
4
Reconstitution and Kinetics of Rhomboid Proteases
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Rhomboid proteases were co-reconstituted with substrates into liposomes using the inducible reconstitution system that we described recently16 (link). Bacterial rhomboid proteases were reconstituted in liposomes formed from an E. coli polar lipid extract, while DmRho4 was reconstituted in liposomes formed from a yeast polar lipid extract or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids (Avanti Polar Lipids). All enzymes were assayed for 1 hour at 37°C, except DmRho4, which was assayed at 25°C for 2–4 hours. APP+Spi7-Flag reaction products were resolved by SDS-PAGE and quantified by anti-Flag western analysis using an Odyssey infrared laser scanner (LiCor Biosciences), while products for the real-time assay using FITC-TatA were quantified using a Synergy H4 Hybrid plate reader (Biotek) scanning once per minute. Proteolysis assays were supplemented with 0.5 mM calcium unless otherwise indicated. For calcium titration experiments, 10 pmol of DmRho4 and 200 pmol FITC-TatA were co-reconstituted into 30 µg yeast liposomes and total calcium was titrated from 0 to 1 mM. For kinetic analysis, 10 pmol of Rho4 was titrated against 15-600 pmol FITC-TatA substrate in the presence or absence of 0.5 mM calcium, initial reaction rates were extracted and fit to a Michaelis-Menten model using Prism software.
5
Cell Viability and Colony Formation Assay
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Cells (100.000/well) were seeded in triplicates on 12-well plates. After transfection cell growth was assessed at each time-point by Trypan Blue exclusion assay using Countess automated cell counter (Invitrogen). Cell viability was evaluated as percentage of live cells in the total cell population. For colony formation assay, 5.000 cells were plated in 6 well plates and grown 10 days. Colonies were fixed with 10% (v/v) methanol for 10min at −20°C, stained with 0.5% Crystal violet solution (made in 25% methanol) for 10min at room temperature and washed with ddH2O for colony visualisation. Colonies were quantified using Odyssey Infrared laser scanner (Li-Cor Biosciences, NE, USA) at Infrared wavelength of 700 nm.
6
Quantitative Western Blot Analysis
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For Western blot analysis, ~2x10 7 cells were harvested from mid-log phase cultures grown at 24°C and total protein was extracted by TCA precipitation as previously published (Keaton et al., 2008) . Electrophoresis and Western blotting were performed as previously described (Bose et al., 2001) . A polyclonal rabbit α-HA epitope antibody preparation was used at 1:2000 dilution (Rockland Immunochemicals). A monoclonal mouse α-Cdc42 antibody preparation (gift from P. Brennwald, UNC) was used at 1:500 dilution (Wu et al., 2015) . Polyclonal goat antibodies against rabbit (Goat anti-Rabbit IgG DyLight 680 conjugated, Rockland Immunochemicals) and mouse (Goat anti-Mouse IgG DyLight 800 conjugated, Rockland Immunochemicals) were used at 1:10,000 dilution as secondary antibodies. Blots were imaged with an ODYSSEY infrared laser scanner (LI-COR Biosciences).
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