Protein lysis of TSC2-null 621-101 cells treated with Fludarabine was done using mammalian protein extraction reagent buffer (Thermo Fisher Scientific, no. 78501) containing protease and phosphatase inhibitors. Lysates were resolved on 4 to 20% Mini-PROTEAN TGX Precast gels (Bio-Rad, no. 4561094) and transferred using a wet electrophoretic transfer unit (Bio-Rad) onto polyvinylidene difluoride membranes (Thermo Fisher Scientific). After 1 hour of blocking with 5% nonfat dry milk, membranes were incubated with primary antibodies at 4°C overnight. Primary antibodies include: phospho-STAT1 (no. 8826), STAT1 (no. 14994), phospho-STAT3 (no. 9145), STAT3 (no. 30835), PBX1 (no. 4342), cleaved caspase-3 (no. 9661), PARP (no. 9532), cyclin D1 (no. 55506), phospho-RIPK1 (no. 44590), and β-actin (no. 3700) from Cell Signaling Technology. Secondary antibody incubation was done using horseradish peroxidase–linked antibodies for 2 hours. Signal was detected with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, no. PI34580).
Mammalian protein extraction reagent buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mammalian protein extraction reagent buffer is a solution designed for the efficient extraction and solubilization of proteins from mammalian cell and tissue samples. It is a balanced buffer system that helps preserve the native structure and function of the extracted proteins.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Wet electrophoretic transfer unit, by Bio-Rad (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3 no 9661, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Ab188570, by Abcam (1 mentions)
Ab3773, by Abcam (1 mentions)
Ab41037, by Abcam (1 mentions)
Ab53015, by Abcam (1 mentions)
Ab15191, by Abcam (1 mentions)
Ab9324, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Yeasen (1 mentions)
Gb11017b, by Wuhan Servicebio Technology (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Fusion solo 7s, by Vilber (1 mentions)
Cdc25a, by Cell Signaling Technology (1 mentions)
Cyclin e2, by Cell Signaling Technology (1 mentions)
4 protocols using mammalian protein extraction reagent buffer
1
Fludarabine-Induced Protein Signaling in TSC2-null Cells
2
Western Blot Analysis of Protein Expression
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After treatment, cells were harvested in a mammalian protein extraction reagent buffer (Thermo Fisher Scientific, IL, USA) on ice. Total proteins were resolved on 8–12% SDS-PAGE gels and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in TBST and then incubated overnight at 4 °C with the first antibodies against IL-6 (Abcam, ab9324, 1:1000), COX2 (Abcam, ab15191, 1:1000), aggrecan (Abcam, ab3773, 1:200), collagen II (Abcam, ab188570, 1:2000), MMP3 (Abcam, ab53015, 1:1000), ATAMTS5 (Abcam, ab41037, 1:250), FBXO6 (Abcam, ab153853, 1:1000) and β-Tubulin (Servicebio, GB11017B, 1; 1000). After washing membranes three times using TBST, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (YEASEN, Shanghai, China). After washing, immunolabeling was detected using the ECL reagent (Thermo Fisher Scientific, IL, USA).
3
Western Blot Protein Extraction and Analysis
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The cells were gently scraped from the culture plates, resuspended in 1000 μL of Mammalian Protein Extraction Reagent buffer (Thermo Fisher Scientific), and shaken for 5 minutes. The samples were then centrifuged at 14,000g for 10 minutes. The supernatants were collected, and the protein concentration was calculated using a Qubit 2.0 fluorometer (Thermo Fisher Scientific). Protein extracts (30 μg per lane) were prepared, run on a 4%–20% Mini-PROTEAN TGX gel (Bio-Rad), and transferred to a 0.45 μm PVDF membrane. The membranes were blocked for 1 hour at room temperature using Blocking One (nacalai tesque), followed by incubation overnight at 4°C with the primary antibodies presented in Supplemental Table 1 . Two secondary antibodies — anti-mouse IgG, HRP-linked whole Ab sheep (GE Healthcare, now Cytiva, NA931-1ML), and anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, 7074S) — were used at a dilution of 1:5000, and the membranes were developed using ImmunoStar LD (Wako) and imaged using the FUSION Solo 7S (Vilber-Lourmat).
4
Apoptosis and Cell Cycle Regulation
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AF and cisplatin were purchased from Sigma-Aldrich (Merck KGaA). Mammalian Protein Extraction Reagent buffer (Thermo Fisher Scientific, Inc.) was used for protein extraction. Protease inhibitor was pur-chased from MilliporeSigma. Primary antibodies against apoptosis-associated proteins, including poly (ADP-ribose) polymerase (PARP; cat. no. 9542S, 1:1,000) and caspase 3 (cat. no. 9662S, 1:1,500), caspase 8 (9746S, 1:1,000), caspase 9 (9502S, 1:1,000) were purchased from Cell Signaling Technology, Inc. Antibodies specific for cell cycle-associated proteins, p21 (2947S, 1:1,000 p21), p27 (cat. no. 2552S, 1:1,000), CDK2 (2546S, 1:1,000), cyclin E2 (cat. no. 4132S, 1:1,000), CDC25A (3652S; 1:1,000), cyclin D1 (2978S, 1:1,000) were purchased from Cell Signaling Technology, Inc. Antibodies for cell-cycle-associated proteins CDK4 (GTX102993, 1:1,000), cyclin A2 (GTX103042, 1:1,000), cyclin E1 (GTX103045, 1:1,000), were purchased from GeneTex. Horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (AB_2307391, 1:3,000, polyclonal) and horse anti-mouse IgG (AB_10015289, 1:3,000, polyclonal), were purchased fromJackson ImmunoReseach, Inc. The antibody against β-actin (loading control; cat. no. sc-47778, 1:7,500) was purchased from Santa Cruz Biotechnology, Inc. N-acetyl-L-cysteine (NAC; cat. no. A7250) and H2DCFDA (D6883) were purchased from Sigma-Aldrich (Merck KGaA).
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