Sc 8327

Manufactured by Santa Cruz Biotechnology

SC-8327 is a laboratory equipment product from Santa Cruz Biotechnology. It is designed for general laboratory use, but a detailed description of its core function is not available at this time.

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Lab products found in correlation

Immobilon p, by Merck Group (2 mentions) Sypro ruby, by Thermo Fisher Scientific (1 mentions) Typhoon imager, by GE Healthcare (1 mentions) Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions) Sc 7482, by Santa Cruz Biotechnology (1 mentions) Tunicamycin, by Merck Group (1 mentions) Phospho 4e bp1, by Abcam (1 mentions) Sc 7160, by Santa Cruz Biotechnology (1 mentions) Antibody against α tubulin, by Merck Group (1 mentions) Rapamycin, by Selleck Chemicals (1 mentions) Sc 200, by Santa Cruz Biotechnology (1 mentions) Nbp1 40256, by Novus Biologicals (1 mentions) Sc 575, by Santa Cruz Biotechnology (1 mentions) Bip 610978, by BD (1 mentions)

3 protocols using sc 8327

SDS-PAGE Protein Separation Assay

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Lysates from Tm and Gb-treated cells or Gadd34 siRNA-treated cells (100 μg total protein) were boiled at 95°C for 1 min in 1X Laemmli sample buffer (60 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.0005% bromophenol blue, and 2% beta-mercaptoethanol). Then, proteins in each sample were separated by 10% SDS-PAGE. Total proteins were visualized using a Typhoon imager (GE Healthcare Life Sciences) after staining with Sypro®Ruby (ThermoFisher). For the Gadd34 Western blot, proteins were transferred onto a PVDF membrane (Milipore Immobilon-P) and then probed with a Gadd34 specific antibody (Santa Cruz Biotechnology, SC-8327).

Dedigama-Arachchige P.M., Acharige N.P, & Pflum M.K. (2018). Identification of PP1-Gadd34 substrates involved in the unfolded protein response using K-BIPS, a method for phosphatase substrate identification. Molecular omics, 14(2), 121-133.

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Pulldown Assay for PP1 and Gadd34

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Lysates (3.5 mg total protein) obtained from Tm and Gb-treated cells were pre-incubated with Gb (50 μm in 0.1 % ethanol) at room temperature for 15 minutes. Lysates (3.5 mg total protein) from cells treated only with Tm were pre-incubated with vehicle (0.1% ethanol) for 15 minutes at room temperature. A fraction of lysates (100 μg) was saved as an input control for gel analysis. The remaining lysates were incubated for 1 hour at 4°C with a PP1 antibody (14 μg, Santa Cruz Biotechnology, SC-7482) with rotation. Next, the lysate-antibody mixture was incubated with Protein A/G agarose beads (20 μL, Santa Cruz Biotechnology, SC-2003) for 1 hour at 4°C. Then, bead-bound proteins were eluted by boiling at 95°C for 5 minutes in 2X Laemmli sample buffer. After separation of eluted proteins by 10% SDS-PAGE, proteins were transferred onto a PVDF membrane (Milipore Immobilon-P) and the levels of PP1 and Gadd34 were monitored by Western blot using specific antibodies (Santa Cruz Biotechnology, SC-7482 for PP1 and SC-8327 for Gadd34).

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Studying ER Stress Signaling Pathways

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Murine recombinant insulin-like growth factor-1 (IGF-1) was purchased from PeproTech (Rocky Hill, NJ). Thapsigargin was obtained from Applichem (St Louis, MO), tunicamycin from Sigma (St Louis, MO) and brefeldin A from TOKU-E (Bellingham, WA). mTOR and PERK inhibitors were purchased from LC Labs (Woburn, MA) (rapamycin), Selleckchem (Houston, TX) (WYE125132, GSK2656157), Abcam (Cambridge, UK) (PP242) and from GlaskoSmith and Kline (Middlesex, UK) (GSK2126458). Antibodies against phospho-PERK (Thr980) (No.3179), PERK (No. 3192), phospho-eIF2α (Ser51) (No. 3597), eIF2α (No.2103), phospho-p70S6K1^thr389 (No. 9234), p70S6K1 (No. 9202) and cleaved-caspase3 (No. 9664) were purchased from Cell Signaling Technology (Beverly, MA). Phospho-4E-BP1 (phosphorylation on Thr45; ab68187), and 4E-BP1 (ab2606) were from Abcam (Cambridge, UK). ATF6 clone 70B1413.1 were from Abcam (Cambridge, UK; ab11909) and Novus Biological (Littleton, CO; NBP1-40256). Antibodies against CHOP (SC-575), ATF4 (SC-200), GADD34 (SC-8327) and XBP1 (SC-7160) came from SantaCruz Biotechnology (Santa Cruz, CA) and BiP (610978) from BD Laboratories^™ (Franklin Lakes, NJ). Antibody against α-tubulin was from Sigma-Aldrich (St. Louis, MO).

Freis P., Bollard J., Lebeau J., Massoma P., Fauvre J., Vercherat C., Walter T., Manié S., Roche C., Scoazec J.Y, & Ferraro-Peyret C. (2017). mTOR inhibitors activate PERK signaling and favor viability of gastrointestinal neuroendocrine cell lines. Oncotarget, 8(13), 20974-20987.

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