For immunoprecipitations (IPs), cells were lysed in lysis buffer (25 mM TrisHCl, pH 7.4, 1% (v/v) Nonidet P-40 (NP-40, AppliChem, Darmstadt, Germany), 150 mM NaCl, protease inhibitors (Complete Protease Inhibitor Cocktail; Roche, Indianapolis, IN), 5% glycerol (AppliChem #A2926) and phosphatase inhibitors (PhosSTOPTM, Roche, Indianapolis, IN), 2 mM sodium orthovanadate) for 20 min with overhead rotation at 4 °C followed by centrifugation (15.000 rpm, 20 min at 4 °C). Postnuclear supernatants were incubated with 3 μg of antibodies coupled to protein A– or protein G–Sepharose beads (GE Healthcare, Solingen, Germany) for 4 h at 4 °C. Beads were washed five times with lysis buffer, bound proteins were eluted by boiling in 3x SDS-sample buffer/150 mM DTT. Eluted proteins were separated by SDS–PAGE and analyzed by Western blotting with near-infrared fluorescence detection (Odyssey Infrared Imaging System Application Software Version 3.0 and IRDye 800CW-conjugated antibodies; LI-COR Biosciences, Bad Homburg, Germany). All IP and Western blot experiments shown in this study are representative for at least three independent experiments.
Odyssey infrared imaging system application software version 3
Manufactured by LI COR
Sourced in Germany
The Odyssey Infrared Imaging System Application Software Version 3.0 is a software package that enables the operation and control of the Odyssey Infrared Imaging System, a laboratory equipment product by LI-COR. The software provides the necessary functionality to acquire, analyze, and manage data generated by the Odyssey Infrared Imaging System.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop, by Roche (2 mentions)
Irdye 800cw conjugated antibody, by LI COR (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Np 40, by AppliChem (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Goat anti rabbit irdye 680, by LI COR (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
βiii tubulin, by Promega (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Protein a or protein g sepharose beads, by GE Healthcare (1 mentions)
6 protocols using odyssey infrared imaging system application software version 3
1
Immunoprecipitation and Western Blot Analysis
2
Cocaine Potentiates HIV Infection in Jurkat Cells
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Jurkat cells (5 × 106 cells) were treated with cocaine for 3 h and infected with replication-competent HIV (HIV Type 1 strain 93/TH/051) for 24 and 48 h. Treatment with inhibitors was done 1 h prior to HIV infection. Thereafter, samples were collected and washed with 1 mL of ice-cold PBS, and we added 100–200μL of RIPA buffer (50 mM Tris pH 8.0, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, and 150 mM NaCl). Cells were resuspended with RIPA and incubated on ice for 30 min. During the 30 min incubation time, cells were vortexed 3 times after every 10 min to ensure complete lysis. The cell lysate was centrifuged at the highest speed for 10 min, and the supernatant was analyzed for the protein concentration using Pierce™ BCA Protein Assay Kit. Protein concentration was normalized, and an equal amount of protein was mixed with 5X Laemmle Sample buffer, heated to 100°C for 10 min. An equal amount of protein was resolved by SDS-PAGE on 12% gel at 120 volts until the dye reached the bottom and was then transferred to nitrocellulose membrane. The membranes were blocked with 3% BSA for 1 h, incubated with primary antibodies at 4°C overnight, and then incubated with Li-cor secondary antibodies (1:15000 dilution) for 1 h at RT. After three rounds of washing with 1X TBST, the blot was detected with Odyssey infrared imaging system application software version 3.0 (Li-cor Bioscience).
3
Quantifying Kidney Probe Intensity in Mice
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Mice were euthanized after in vivo microCT–FMT imaging by isoflurane overdose. Blood samples were harvested by cardiac puncture, transferred to EDTA coagulation vials and centrifuged at 4600 rpm for 10 min to collect plasma. Next, kidneys were excised, emersion-fixated in formalin and assessed for ex vivo tissue epifluorescence using the FMT system and the Odyssey® CLx imaging system (LI-COR® Biosciences, Lincoln, NE, USA). Probe intensity was quantified with the Odyssey infrared imaging system application software version 3.0 (LI-COR® Biosciences, Lincoln, NE, USA). In order to quantify the probe intensity per kidney, the integrated intensity was divided by the shape area, resulting in counts/mm2 per kidney. A separate group of Ercc1d/− and WT mice was sacrificed, kidneys were excised, snap frozen in liquid nitrogen and stored at −80 °C.
4
Western Blot Analysis of CDK5 and p35 in Hippocampus
5
Detailed Protein Quantification Protocol
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All measured values are shown as mean value with standard deviation. RNA concentrations were determined using the NanoDrop 2000c spectrophotometer and the associated software.
For evaluation of In-Cell Western ® and western blotting, the plates and blots were analyzed with the Odyssey Infrared Imager and the Odyssey Infrared Imaging System Application Software Version 3.0 by LI-COR ® Biosciences. The statistical significance of differences between the values of the controls and treated samples was determined by ANOVA using GraphPad Prism 8 and Excel. In each case, *p<0.05 and **p<0.01 were considered statistically significant. ANOVA results are presented as [F(degrees of freedom between treatment group, degrees of freedom within treatment group)=F statistic, p-value]. Tukey post-hoc tests were performed on statistically significant ANOVAs to determine which of the treatment groups were significantly different from each other.
For evaluation of In-Cell Western ® and western blotting, the plates and blots were analyzed with the Odyssey Infrared Imager and the Odyssey Infrared Imaging System Application Software Version 3.0 by LI-COR ® Biosciences. The statistical significance of differences between the values of the controls and treated samples was determined by ANOVA using GraphPad Prism 8 and Excel. In each case, *p<0.05 and **p<0.01 were considered statistically significant. ANOVA results are presented as [F(degrees of freedom between treatment group, degrees of freedom within treatment group)=F statistic, p-value]. Tukey post-hoc tests were performed on statistically significant ANOVAs to determine which of the treatment groups were significantly different from each other.
6
Immunoprecipitation Protocol with Detailed Lysis Conditions
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For immunoprecipitations, cells were lysed in lysis buffer (25 mM TrisHCl, pH 7.4, 1% (v/v) Nonidet P-40 (NP-40, AppliChem, Darmstadt, Germany), 150 mM NaCl, protease inhibitors (Complete Protease Inhibitor Cocktail; Roche, Indianapolis, IN), 5% glycerol ()AppliChem #A2926) and phosphatase inhibitors (PhosSTOP TM , Roche, Indianapolis, IN), 2 mM sodium orthovanadate) for 20 min with overhead rotation at 4°C followed by centrifugation (15.000 rpm, 20 min at 4°C). Postnuclear supernatants were incubated with 3 μg of antibodies coupled to protein A-or protein G-Sepharose beads (GE Healthcare, Solingen, Germany) for 4 h at 4°C. Beads were washed five times with lysis buffer, bound proteins were eluted by boiling in 3x SDS-sample buffer/150 mM DTT. Eluted proteins were separated by SDS-PAGE and analyzed by Western blotting with near-infrared fluorescence detection (Odyssey Infrared Imaging System Application Software Version 3.0 and IRDye 800CW-conjugated antibodies; LI-COR Biosciences, Bad Homburg, Germany).
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