Cells and tissues were collected using a 5% SDS, 100mM Tris pH=7 solution and incubated on ice for 15 min, during which time the samples were passed through a large guage needle several times and vortexed every 5min. Samples were then spun at 14,000 RPM to remove debris and the supernatant was mixed 1:1 with 2x laemmli buffer. Samples were boiled for 20min before being centrifuged at 14,000 RPM for 1min and loaded into a BioRad Criterion 4–20% gel. After transfer to PDVF membrane and blocked (5% dehydrated milk) for 2hr at RT, membranes were incubated overnight with 1:500 dilution of ORF1p or 1:10,000 β-tubulin in 2.5% blocking buffer. Membranes were washed 3x with TBST for 10 min each before secondary antibody in TBST was added for 2hr incubation at RT. Membranes were washed and then imaged.
Criterion 4 20 gel
Manufactured by Bio-Rad
The Criterion 4–20% gel is a pre-cast polyacrylamide gel used for protein separation and analysis. The gel has a gradient of 4% to 20% acrylamide, which allows for the effective separation of a wide range of protein molecular weights. This product is designed for use in electrophoresis applications.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay kit, by Bio-Rad (2 mentions)
Imagequant las 4000 system, by GE Healthcare (2 mentions)
Laemmli buffer, by Bio-Rad (2 mentions)
Ne per kit, by Thermo Fisher Scientific (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Sirt6, by Cell Signaling Technology (1 mentions)
Rabbit anti flag, by Merck Group (1 mentions)
Goat anti mouse irdye 680, by LI COR (1 mentions)
Goat anti rabbit irdye 800, by LI COR (1 mentions)
Rabbit anti cas9, by Abcam (1 mentions)
Mouse anti tubulin, by Merck Group (1 mentions)
5 protocols using criterion 4 20 gel
1
Cell Protein Extraction and Western Blot
2
Comprehensive Protein Analysis Workflow
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Cells and reactions were collected using a 2x Laemmli solution and incubated on ice for 15 min, during which time the samples were passed through a large gauge needle several times and vortexed every 5 min. Samples were then spun at 14,000 RPM to remove debris, and the supernatant was transferred to a new tube. Samples were heated in boiling water for 20 min before being centrifuged at 14,000 RPM for 1 min and loaded into a BioRad Criterion 4–20% gel. After transfer to PDVF membrane and blocked (5% dehydrated milk) for 2 h at RT, membranes were incubated overnight antibodies in 2.5% blocking buffer at 4°C. Membranes were washed 3x with TBST for 10 min each before secondary antibody in 1x TBST was added for 2 h incubation at RT. Membranes were washed 3x with TBST for 10 min and then imaged.
The following antibodies were used: H3 (Abcam ab500)‐1:5,000, H3K9ac (Abcam ab4441)‐1:1,000, H3K18ac (Abcam ab1191)‐1:1,000, β‐tubulin (Abcam ab6046)‐1:10,000, SIRT6 (Cell Signaling #12486)‐1:1,000, Lamin A/C‐1:1,000 (Abcam ab108595, Millipore 05–714), γH2AX (Millipore 05–636), PARP1(Abcam ab227244)‐1:1,000, Vimentin‐1:1,000 (Abcam ab92547); TAF15‐1:2,000 (Abcam ab134916); mADPr 1:500 (AbD33204 and AbD33205)(Bonfiglio et al, 2020 (link)).
The following antibodies were used: H3 (Abcam ab500)‐1:5,000, H3K9ac (Abcam ab4441)‐1:1,000, H3K18ac (Abcam ab1191)‐1:1,000, β‐tubulin (Abcam ab6046)‐1:10,000, SIRT6 (Cell Signaling #12486)‐1:1,000, Lamin A/C‐1:1,000 (Abcam ab108595, Millipore 05–714), γH2AX (Millipore 05–636), PARP1(Abcam ab227244)‐1:1,000, Vimentin‐1:1,000 (Abcam ab92547); TAF15‐1:2,000 (Abcam ab134916); mADPr 1:500 (AbD33204 and AbD33205)(Bonfiglio et al, 2020 (link)).
3
Western Blot Analysis of Cas9 Expression
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Cells were collected and lysed in 2 × reducing sample buffer, and then incubated at 98°C for 15 min. All samples collected from an individual experiment were run on the same gel to compare protein expression levels as accurately as possible. Samples were separated by a Criterion 4–20% gel (No. 5671095; Bio-Rad), and then transferred to PVDF membranes. Antibody incubation was conducted as previously reported.42 (link) Primary antibodies used include rabbit anti-Cas9 (No. ab189380, 1:5000; Abcam), mouse anti-tubulin (No. T5168, 1:10,000; Sigma-Aldrich), and rabbit anti-FLAG (No. F7425, 1:5000; Sigma-Aldrich). Secondary antibodies used were goat anti-rabbit IRdye800 (No. 925-32211, 1:10,000; LI-COR) and goat anti-mouse IRdye680 (No. 926-69020, 1:10,000; LI-COR).
4
TDP-43 Subcellular Fractionation and Immunoblotting
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24–48 h post-transfection with V5-tagged TDP-43 and RRM mutants, TDP-43 CRISPR KO HeLa cells were lysed for N/C fractionation and SDS-PAGE with the NE-PER kit (ThermoFisher) according to the manufacturer’s instructions. Total protein concentration was measured using the DC Protein Assay kit (Bio-Rad). Nuclear (5 μg total protein) and cytoplasmic (10 μg total protein) fractions were boiled in Laemmli buffer (Bio-Rad), run on Criterion 4–20% Gels (Bio-Rad), and transferred to nitrocellulose membranes using a TransBlot Turbo system (BioRad). Membranes were blocked with 5% non-fat milk in TBS-Tween and probed by sequential incubation in primary antibody overnight at 4°C. Detection was via HRP-conjugated secondary antibodies/chemiluminescence using an ImageQuant LAS 4000 system (GE). Band intensities were measured using ImageQuant software.
5
TDP-43 Subcellular Fractionation and Immunoblotting
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