Polyacrylamide sds gels

Manufactured by Bio-Rad

Polyacrylamide SDS gels are laboratory equipment used for the separation and analysis of proteins based on their molecular weight. They provide a physical matrix for the electrophoretic separation of proteins in the presence of the denaturing agent sodium dodecyl sulfate (SDS).

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Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) A5441, by Merck Group (1 mentions) Mouse anti β actin c4, by Santa Cruz Biotechnology (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) α tubulin, by Merck Group (1 mentions) Odyssey infrared imager, by LI COR (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions)

3 protocols using polyacrylamide sds gels

Validating HIF Protein Expression in Ovarian Cancer Cells

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Western blots were performed on A2780s and TOV112D ovarian cancer cells to validate the expression of HIF at the protein level at different time points. Cells grown in hypoxic chambers were frozen at the time points mentioned in the results section and harvested all together by lysing in Radioimmunoprecipitation assay buffer (RIPA buffer) containing 1 × protease inhibitor cocktail (Sigma-Aldrich). For each sample, a 30 μg aliquot was loaded onto pre-cast polyacrylamide-SDS gels from BioRAd that were then transferred onto nitrocellulose. Membranes were incubated with primary antibodies against HIF-1α (#610958, BD Biosciences), Tubulin (#2144, CST) or β-Actin (A5441, Sigma, 1:4000) overnight at 4° C, followed by fluorescent-conjugated secondary antibodies (IRDye® 800CW Goat anti-MouseIgG and IRDye® 8680CW Goat anti-rabbit IgG). An Odyssey chemiluminescence-fluorescence system was used for protein detection. Proteins detected ran at the expected molecular weights, as verified using molecular weight standard markers.

Pressley M., Gallaher J.A., Brown J.S., Tomaszewski M.R., Borad P., Damaghi M., Gillies R.J, & Whelan C.J. (2021). Cycling hypoxia selects for constitutive HIF stabilization. Scientific Reports, 11, 5777.

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Western Blotting for SNAP25 Cleavage

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Full western blotting methodology is available in supplemental methods. Briefly, to measure onabotA cleavage of SNAP25 in TG neurons cultured from wild-type mice, treated cultured TG neurons were lysed using modified RIPA buffer, sonicated, and centrifuged to be used for western blot analysis. Protein was quantified for equal sample loading (BCA Protein Assay Kit, ThermoFisher Scientific), samples were electrophoresed on polyacrylamide SDS gels (Biorad), and proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Blots were blocked in buffer for one hour at room temperature (RT) before overnight incubation at 4°C with the following primary antibodies in blocking buffer: mouse anti-β-Actin (C4) (Santa Cruz Biotechnology, sc-47778) and recombinant human anti-SNAP25₁₉₇ (Allergan plc, Ab632), a highly selective antibody for the cleaved epitope of SNAP25 (33 (link)). Blots were washed, incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Jackson ImmunoResearch), and developed by enhanced chemiluminescence.

Morgenstern J.P., Griffith I.J., Brauer A.W., Rogers B.L., Bond J.F., Chapman M.D, & Kuo M.C. (1991). Amino acid sequence of Fel dI, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning.. Proceedings of the National Academy of Sciences of the United States of America, 88(21), 9690-9694.

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Western Blot Analysis of RAS Signaling

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Protein lysates were resolved on 7.5, 4–12, or 8–16% polyacrylamide SDS gels (Bio-Rad), transferred onto nitrocellulose membranes (Bio-Rad) using standard wet-transfer procedures, and incubated with primary antibodies as indicated. All immunoblot assays were visualized using a LI-COR Odyssey infrared imager. The following antibodies were used: KRAS (Proteintech Group 12063-1-AP), RAS (CST 3965), RAS (clone 10, EMD Millipore 05-516), pERK (CST 4370), ERK (CST 9102), pAKT (S473, CST 4060), a-tubulin (Sigma Aldrich, clone DM1A, T9026), NRF2 (CST 12721), and NRF2 (R&D Systems AF3925) (CST: Cell Signaling Technologies). Secondary anti-rabbit and anti-mouse IRDye antibodies were from LI-COR Biosciences.

Kim E., Ilic N., Shrestha Y., Zou L., Kamburov A., Zhu C., Yang X., Lubonja R., Tran N., Nguyen C., Lawrence M.S., Piccioni F., Bagul M., Doench J.G., Chouinard C.R., Wu X., Hogstrom L., Natoli T., Tamayo P., Horn H., Corsello S.M., Lage K., Root D.E., Subramanian A., Golub T.R., Getz G., Boehm J.S, & Hahn W.C. (2016). Systematic functional interrogation of rare cancer variants identifies oncogenic alleles. Cancer discovery, 6(7), 714-726.

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