Cells were harvested in lysis buffer (1% Triton X-100, 150mM NaCl, 20mM Tris pH 8, 1mM EDTA, 1mM EGTA, 0.1% SDS, 1mM beta-glycerol-phosphate, 1mM Na-pyrophosphate) with fresh protease and phosphatase inhibitors on ice for 10 min before pelleting debris for 10 min at 13,000 rpm. Lysate concentration was measured using Bradford assay (BioRad) against a standard of bovine serum albumin (BSA), and samples were prepared with 4X SDS buffer (BioRad) and 10X dithiothreitol (DTT, Thermo Fisher Scientific) and boiled at 95°C for 5min. Equal amounts of protein were separated by SDS-PAGE on 4-15% gradient Mini-Protean-TGX (BioRad), transferred to nitrocellulose membranes (Pall 66485) followed by appropriate overnight primary and 1h secondary antibody incubations (see key resources table ). Following Clarity Western enhanced chemiluminescence (ECL) detection (BioRad), blots were imaged using ChemiDoc platform (BioRad). Densitometry was quantified using ImageJ and normalized to beta-Actin or GAPDH loading controls.
Chemidoc platform
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc platform is a laboratory imaging system designed for the detection and analysis of chemiluminescent and fluorescent signals. It provides high-quality image capture and quantification capabilities for a variety of applications, including Western blotting, gel documentation, and protein and nucleic acid detection.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Gradient gel, by Bio-Rad (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Mouse anti ppm1d f 10, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Horseradish peroxidase linked secondary antibody, by Abcam (1 mentions)
Pvdf sheet, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Mouse anti gapdh mab374, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti γ h2ax ser139, by Cell Signaling Technology (1 mentions)
Hybond c membrane, by Cytiva (1 mentions)
Sc 32736, by Santa Cruz Biotechnology (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
6 protocols using chemidoc platform
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis in H9C2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples from H9C2 cells were extracted with RIPA buffer (Thermo Fisher Scientific, USA) supplemented with protease and phosphatase inhibitors. The BCA kit (Thermo Fisher Scientific, USA) measured protein concentrations. SDS‐PAGE resolved proteins, which were then blotted onto PVDF sheets (Millipore, USA). These membranes were subsequently probed using primary antibodies targeting FLRT3, SMAD4, ANP, BNP, Bcl‐2, Bax, LC3I, LC3II, SCN5A, KCNIP2, KCND2 (all 1:1000, Abcam) and β‐actin (1:5000, Cell Signalling Technology) for normalization. Following primary incubation, blots were exposed to horseradish peroxidase‐linked secondary antibodies (1:5000, Abcam). Detection was accomplished via the ECL kit (Thermo Fisher Scientific) and visualized on a ChemiDoc platform (Bio‐Rad, USA).
3
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or minced mouse tissue were lysed with 1x RIPA buffer supplemented with the Halt Protease and Phosphatase inhibitor cocktail (ThermoFisher) for 30 minutes at 4°C. Protein concentration was quantified using the Pierce BCA protein assay kit (Thermo Fisher), and lysates were then boiled at 95C in 1x Laemmli (Bio-Rad) for 5 mins. The proteins were separated by SDS-PAGE on 4%–15% gradient gels (Bio-Rad), and transferred onto nitrocellulose membranes (Bio-Rad). For the blot in Figure 3 A, duplicate sets of samples were run for parallel blotting. After 1 hour of blocking, membranes were incubated overnight at 4°C with the following primary antibodies: mouse anti-PPM1D (F-10, Santa Cruz, 1:1000, for detection of human PPM1D), rabbit anti-p53 ser15 (#9284, Cell Signaling, 1:1000), rabbit anti-total p53 (#9282, Cell Signaling, 1:1000), rabbit anti-γH2AX ser139 (#2577, Cell Signaling, 1:1000), rabbit anti-total H2AX (#2595, Cell Signaling, 1:1000), mouse anti-GAPDH (MAB374, Millipore, 1:2000), rabbit anti-PPM1D (D4F7, Cell Signaling, 1:1000, for detection of mouse PPM1D). This was followed by secondary antibody incubation with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (Santa Cruz), and imaging on the Bio-Rad ChemiDoc platform.
4
Enrichment and Quantification of TOP1-DNA Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TOP1 protein–DNA complexes (TOP1cc) were purified using caesium chloride density gradients. Cells (2 × 106) were lysed in 1% sarcosyl, 8 M guanidine HCl, 30 mM Tris pH 7.5 and 10 mM EDTA. Cell lysates were incubated at 70°C for 15 min to remove all non-covalently bound proteins from DNA. Cell lysates were then loaded on a caesium chloride density (CsCl) step gradient (5 ml total volume) and centrifuged at 75 600 × g at 25°C for 24 h to separate free proteins from DNA. Ten consecutive 0.5 ml fractions were collected and slot blotted onto Hybond-C membrane (Amersham). To ensure equal DNA loading, the DNA concentration in each extract was determined fluorimetrically using PicoGreen (Molecular Probes/Invitrogen). Covalent TOP1–DNA complexes were then detected by immunoblotting with anti-TOP1 antibodies (sc-32736, Santa Cruz) and visualised by chemiluminescence. Fractions enriched for TOP1cc were pooled and subjected to serial dilution followed by band quantifications using the BioRad ChemiDoc platform.
5
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or minced mouse tissue were lysed with 1 x RIPA buffer supplemented with the Halt Protease and Phosphatase inhibitor cocktail (ThermoFisher) for 30 min. Following, protein concentration was quantified, and lysates were then boiled at 95 C in 1 x Laemmli (Bio-Rad) for 5 min. The proteins were separated by SDS-PAGE on 4–15% gradient gels (Bio-Rad), and transferred onto nitrocellulose membranes (Bio-Rad). After 1 hr of blocking in 3% skim milk, membranes were incubated overnight with the following primary antibodies: anti- actin (Santa Cruz), anti-Dnmt3a (Cell signaling), anti-p-HSL (Cell Signaling), anti-STAT3 (Cell Signaling), anti-p-STAT3 (Cell Signaling). This was followed by secondary antibody incubation with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (Santa Cruz), and imaging on the Bio-Rad ChemiDoc platform. Antibodies used: β-Actin Antibody (Cell Signaling, Cat$4,967 S), phosphor-HSL(Ser660) (Cell Signaling. Cat #4,126 S).
6
Immunoblot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with 1x RIPA buffer supplemented with Halt Protease and Phosphatase inhibitor cocktail (Thermofischer) for 1 hour at 4°C. Protein concentration was quantified using the Pierce BCA protein assay kit (Thermofischer) and boiled at 95°C in 1x Laemmli (Biorad) for 7 minutes. The samples in which mitochondrial proteins were probed were not boiled, as boiling can cause signal reduction. Instead, samples were warmed to 37°C for 30 minutes prior to loading. The proteins were separated by SDS-PAGE on 4–15% gradient gels (Biorad) and transferred on to PVDF membranes using the iBlot Dry Blotting system (Thermofisher). Membranes were incubated for 1 hour at room temperature in 5% milk in Tris-buffered saline solution with Tween-20 (TBST). After washing, the membranes were incubated overnight at 4°C with the following primary antibodies: mouse anti-PPM1D (F-10, Santa Cruz, 1:1000), mouse anti-GAPDH (MAB374, Millipore, 1:200), mouse total OXPHOS Human antibody cocktail (ab110411, Abcam, 1:1000), mouse anti-Vinculin (V9131, Sigma Aldrich, 1:2000). The following day, membranes were washed twice with TBST and incubated for 1 hour with HRP-linked anti-rabbit IgG or anti-mouse IgG (Cell Signaling, 1:5000 – 1:10,000) at room temperature. Blots were imaged on the Bio-Rad ChemiDoc platform.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!