The cells were scrapped in the PBS and lysed by using a NP-40 lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, and 1% NP-40) supplemented with antiproteases and antiphosphatases (Roche). 25–40 μg of total protein extracts was separated on 10% SDS-polyacrylamide gel and electrically blotted to nitrocellulose membrane. The proteins were detected using a buffer containing 0.1% Tween 20 and 5% milk and incubated overnight at 4°C with specific primary antibodies and were visualized with IRDye 800 or IRDye700 (Rockland) as secondary antibodies. Quantification was realized using the Odyssey Infrared Imaging System (Li-COR).
Anti proteases and anti phosphatases
Manufactured by Roche
Anti proteases and anti phosphatases are laboratory reagents used to inhibit the activity of proteases and phosphatases, respectively, in biological samples. They are commonly used in various research applications to preserve the integrity of proteins and other biomolecules during sample preparation and analysis.
Automatically generated - may contain errors
3 protocols using anti proteases and anti phosphatases
1
Western Blot Protein Analysis
2
Reverse Phase Protein Array Analysis
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Cell lysates were prepared from three biological replicates in 50mM Hepes pH7.4, 1% Triton X-100, 150mM NaCl, 1.5mM MgCl2, 1mM EGTA, 10% Glycerol containing anti proteases and anti phosphatases (Roche) and sent for analysis at the Reverse Phase Protein Array Core Facility at MD Anderson. Relative protein levels are obtained by interpolating several sample dilutions into a standard curve and then normalized for protein loading. The scatter plot was generated by graphing the difference between irradiated and non-irradiated averages of triplicate samples normalized and transformed to linear values. Included are only the proteins that yielded a significant difference (paired t-test, p<0.05) in HBEC3-KT and/or U2OS cell lysates. Heat maps were generated with normalized log2 median centered data for each marker employing Morpheus software (Broad Institute).
3
Reverse Phase Protein Array Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared from three biological replicates in 50mM Hepes pH7.4, 1% Triton X-100, 150mMNaCl, 1.5mM MgCl 2 , 1mMEGTA, 10% Glycerol containing anti proteases and anti phosphatases (Roche) and sent for analysis at the Reverse Phase Protein Array Core Facility at MD Anderson. Relative protein levels are obtained by interpolating several sample dilutions into a standard curve and then normalized for protein loading. The scatter plot was generated by graphing the difference between irradiated and non-irradiated averages of triplicate samples normalized and transformed to linear values. Included are only the proteins that yielded a significant difference (paired t-test, p<0.05) in HBEC3-KT and/or U2OS cell lysates. Heat maps were generated with normalized log2 median centered data for each marker employing Morpheus software (Broad Institute).
105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprint this version posted June 29, 2020. ; https://doi.org/10.1101/2020.06.29.177352 doi: bioRxiv preprint IR induces replication stress
105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprint this version posted June 29, 2020. ; https://doi.org/10.1101/2020.06.29.177352 doi: bioRxiv preprint IR induces replication stress
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