Cells were lysed in presence of proteinase and phosphatase inhibitors (Complete mini and PhoSTOP, Roche). Protein amounts were assessed via BCA assay (Sigma). Proteins were separated in precast Novex or BOLT 4–12% Bis–Tris gradient gels (Life Technologies) and blotted onto nitrocellulose membranes. Blocking was carried out with 5% milk in TBST. Antibodies (Xpg (sc-12558, Santa Cruz Biotechnology), p53 (#2524, Cell Signalling), phospho-p53 (Ser15, #12571, Cell Signalling), p21 (F-5, Santa Cruz Biotechnology), Gapdh (ACR001PT, Acris), beta-Actin (Sigma)) were detected with secondary IR-Dye coupled secondary antibodies in a Li-Cor Odyssey SA Infrared Western blot detection system. Quantification was carried out using Li-Cor software.
Bolt 4 12 bis tris gradient gels
Manufactured by Thermo Fisher Scientific
The Bolt 4–12% Bis-Tris gradient gels are electrophoresis gels designed for the separation and analysis of proteins. These gels feature a 4-12% gradient of bis-tris acrylamide, which allows for the effective separation of a wide range of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Laemmli sample buffer, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca assay, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Complete mini, by Roche (1 mentions)
Phostop, by Roche (1 mentions)
Epiquick total histone extraction kit, by Epigentek (1 mentions)
Anti h3k9me3, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west atto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated gapdh, by Cell Signaling Technology (1 mentions)
Anti ha tag, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
6 protocols using bolt 4 12 bis tris gradient gels
1
Protein Characterization via Western Blot
2
Histone Extraction and Western Blotting
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The EpiQuick Total Histone Extraction kit (Epigentek) was used to isolate histones. Histone lysates were size separated on Bolt 4–12% Bis-Tris gradient gels (Life Technologies), transferred to polyvinylidene difluoride membrane and detected using standard western blotting technique. GATA2 protein was detected by lysing cells in RIPA denaturing buffer supplemented with protease inhibitors followed by detection using a Wes capillary electrophoresis system (Protein Simple) or western blotting on nitrocellulose membrane. Antibodies used for western blotting: Anti-GATA2 [clone EPR2822(2)] (Abcam), anti-ACTB [clone 1E35] (Cell Signaling Technology), anti-H3K9/14 [catalog number 17–615] (EMD Millipore), anti-H3K56ac [clone EPR996Y] (Abcam), anti-H3K79 [catalog number 07–750] (EMD Millipore), anti-H2BK5 [catalog number 2574] (Cell Signaling Technology), anti-H3K9me3 [catalog number 07–442] (EMD Millipore), and anti-total-H4 [clone 62-141-13] (EMD Millipore).
3
Western Blot Protein Analysis
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Cells were lysed in Pierce RIPA buffer (Thermo Fisher Scientific) supplemented with 1% HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Extracted protein samples were normalized using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), fractionated on Bolt 4%–12% Bis-Tris gradient gels (Invitrogen), and transferred to 0.2-μm PVDF membranes (GE Healthcare). Primary antibodies and HRP-conjugated secondary antibodies are shown in Supplemental Table 5 . SuperSignal West Femto or SuperSignal West Atto chemiluminescent substrate (Thermo Fisher Scientific) was used for imaging. Fiji (ImageJ) was used to analyze band intensity.
4
Western Blot Protein Detection
5
SDS-PAGE Protein Separation
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A total of 20 mg of protein from each sample was mixed with Laemmli sample buffer (Bio-Rad) and β-mercaptoethanol before being boiled at 95° for 10 min, spun briefly to collect volume, and loaded into Bolt 4–12% Bis-Tris gradient gels (Invitrogen). Loaded gels were initially electrophoresed at 80 mV for the lowest percentage gradient of the gel, followed by 120 mV for the remainder of the gel. The gels were then submerged in Coomassie blue staining overnight and destained briefly the following day to visualize protein banding (Supplemenatary Figure 5 ).
6
SDS-PAGE Protein Visualization
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20 ug of protein from each sample was mixed with Laemmli sample buffer (BioRad) and β-mercaptoethanol before being boiled at 95° for 10 minutes, spun briefly to collect volume and loaded into Bolt 4–12% Bis-Tris gradient gels (Invitrogen). Loaded gels were initially electrophoresed at 80 mV for the lowest percentage gradient of the gel followed by 120 mV for the remainder of the gel. Gels were then submerged in Coomassie blue staining overnight and destained briefly the following day to visualize protein banding (Supplemental Figure 4 ).
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