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Denaturing polyacrylamide gels

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Denaturing polyacrylamide gels are a type of electrophoresis gel used for the separation and analysis of proteins. They are composed of polyacrylamide and function by separating proteins based on their molecular weight under denaturing conditions.

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Lab products found in correlation

β actin antibody, by Merck Group (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) Anti hk2, by Cell Signaling Technology (1 mentions) Anti puma, by Cell Signaling Technology (1 mentions) Anti bcl 2, by Santa Cruz Biotechnology (1 mentions) Anti hk1, by Cell Signaling Technology (1 mentions) Anti noxa, by Enzo Life Sciences (1 mentions) Anti bim, by Cell Signaling Technology (1 mentions) Enhanced chemiluminescence, by Bio-Rad (1 mentions) Rabbit anti galnac t5, by Abcepta (1 mentions) Hamster anti muc1 ct ct2, by Abcam (1 mentions) Mouse anti β actin, by Merck Group (1 mentions) Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase conjugated anti human igg, by Thermo Fisher Scientific (1 mentions) Trans blot turbo, by Bio-Rad (1 mentions)

Close spelling variants of this product

Bis-Tris XT denaturing 10% SDS polyacrylamide gels 4–20% denaturing Gel Polyacrylamide gel

4 protocols using denaturing polyacrylamide gels

1

Quantitative Western Blotting of Retinal Proteins

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Retina and proximal optic nerve protein samples were analyzed by quantitative Western blotting that followed a methodology similar to previously described.19 (link),22 (link)–24 (link, link) Briefly, protein samples were separated on denaturing polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (Bio-Rad). Following a blocking step, membranes were probed with a phosphorylation site–specific primary antibody to NF-κB subunit, p65 (phospho-Ser536) (1:500; Abcam). After a second blocking step, membranes were incubated with a secondary antibody conjugated with horseradish peroxidase (1:2000; Sigma-Aldrich Corp., St. Louis, MO, USA). Immunoreactive bands were visualized by enhanced chemiluminescence using commercial reagents (GE Healthcare, Pittsburgh, PA, USA). A beta-actin antibody (1:1000; Sigma-Aldrich Corp.) was used to reprobe the stripped immunoblots for loading and transfer control.
Yang X., Hondur G, & Tezel G. (2016). Antioxidant Treatment Limits Neuroinflammation in Experimental Glaucoma. Investigative Ophthalmology & Visual Science, 57(4), 2344-2354.
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2

Western Blotting of Cell Signaling Proteins

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Whole-cell extracts were prepared using 8 M UREA 0.5% Triton-X lysis buffer and 10–75 μg total protein was loaded onto denaturing polyacrylamide gels (Bio-Rad). Blots were incubated with the primary antibodies in TBS 0.05% Tween-20/ 5% milk or 5% BSA overnight at 4 °C, incubated with HRP coupled secondary goat anti-rabbit and goat anti-mouse antibody (Cell signaling) at 1:5,000 for 1 h at room temperature. Primary antibodies used were anti-HK1 (Cell Signaling, #2024, 1:1000), anti-HK2 (Cell signaling, #2867, 1:1000), anti-HK3 (Thermo Fisher Scientific, #PA5-29304, 1:500), anti-yH2AX Ser139 (Cell Signaling, #2577, 1:1000), anti-cleaved Caspase-3 (Cell Signaling, #966, 1:1000 Milk/TBS-T), anti-Puma (Cell Signaling, #12450, 1:1000 BSA/TBS-T), anti-Noxa (Enzo Life Sciences, ALX-804-408-C100, 1:1000 Milk/TBS-T), anti-BCL-2 (Santa Cruz, sc-509, 1:1000 BSA/TBS-T) and anti-BIM (Cell Signaling, #2933, 1:1000 BSA/TBS-T). Cytoplasmic and nuclear fractionation was performed as described before [25 (link)]. Uncropped western blots are shown in the supplement.
Seiler K., Humbert M., Minder P., Mashimo I., Schläfli A.M., Krauer D., Federzoni E.A., Vu B., Moresco J.J., Yates JR 3.r.d., Sadowski M.C., Radpour R., Kaufmann T., Sarry J.E., Dengjel J., Tschan M.P, & Torbett B.E. (2022). Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions. Cell Death & Disease, 13(5), 448.
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3

Protein Isolation and Analysis in Pancreatic Cancer Cells

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NP-40 cell lysis buffer containing protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) was used to isolate proteins from pancreatic cancer cells. Equal concentrations of protein lysates (50 μg) were resolved using 4–20% denaturing polyacrylamide gels (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene fluoride (PVDF) membrane (Millipore EMD, Billerica, MA, USA) and blocked with 5% powdered milk in Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 (TBS-T). The following primary antibodies were used to probe the membrane for the protein expressions: rabbit anti-GalNAc-T5 (Abgent, San Diego, CA, USA, 1:1000), hamster anti-MUC1-CT (CT2, Abcam, USA, 1:1000), and mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA, 1:5000). After incubating with respective secondary antibodies, protein-antibody complex was detected using enhanced chemiluminescence (Bio-Rad, USA).
Caffrey T., Sagar S., Thomas D., Lewallen M.E., Hollingsworth M.A, & Radhakrishnan P. (2019). MUC1 Negatively Regulates GalNAc Transferase 5 Expression in Pancreatic Cancer. FEBS letters, 593(19), 2751-2761.
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4

Western Blot Analysis of Viral Proteins

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All proteins and pseudovirions were run on 4–20% denaturing polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane using Trans-blot Turbo (Bio-Rad, Hercules, CA). Membranes were rinsed with PBST, blocked with 5% skim milk in PBST for 1 h followed by probing of GP and p24 with monoclonal antibody (mAb) H3C8 at a dilution of 1:1000, [60 (link)] and mouse mAB B1217M at a dilution of 1:2000 (Genetex, Irvine, CA), respectively. The mAb H3C8 was humanized by cloning its variable heavy and light chain fragments in human IgG expression vectors obtained from Dr. Michel Nussenzwieg (The Rockefeller University). Membranes were washed three times with PBST, incubated with horseradish peroxidase-conjugated anti-human IgG (Invitrogen, ThermoFisher, Waltham, MA) and anti-mouse IgG (ThermoFisher, Waltham, MA) for 1 h at room temperature and developed with SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific, Waltham, MA).
Jain A., Govindan R., Berkman A.R., Luban J., Díaz-Salinas M.A., Durham N.D, & Munro J.B. (2023). Regulation of Ebola GP conformation and membrane binding by the chemical environment of the late endosome. PLOS Pathogens, 19(12), e1011848.
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