Sc 23957

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Sc-23957 is a laboratory product offered by Santa Cruz Biotechnology. This equipment is designed for use in scientific research and analysis. The core function of Sc-23957 is to facilitate specific experimental procedures, though details on its intended use are not provided.

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Lab products found in correlation

Ab200828, by Abcam (2 mentions) Igg antibody 5145s, by Cell Signaling Technology (2 mentions) 4 6 diamidino 2 phenylindole 4083s, by Cell Signaling Technology (2 mentions) Sa00003 1, by Proteintech (2 mentions) Sa00007 2, by Proteintech (2 mentions) L7543, by Merck Group (1 mentions) Anti p62, by Merck Group (1 mentions) Mg132 c2211, by Merck Group (1 mentions) P0067, by Merck Group (1 mentions) Anti β actin ap0060, by Bioworld Technology (1 mentions) Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies, by Jackson ImmunoResearch (1 mentions) Nocodazole m1404, by Merck Group (1 mentions) Anti lc3, by Merck Group (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Protein g sepharose 4 fast flow beads, by GE Healthcare (1 mentions) Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions) Df8160, by Affinity Biosciences (1 mentions)

6 protocols using sc 23957

Antibody Selection and Characterization

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Antibodies and their manufacturers were as follows: anti-β-actin (AP0060; Bioworld Technology, St. Louis Park, MN, USA), anti-α glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000) (AT0002; CMCTAG, Dover, DE, USA), anti-GFP (1:5000) (AT0028; CMCTAG), anti-FLAG (1:5000) (AT0022; CMCTAG), anti-LC3 (1:3000) (L7543; Sigma-Aldrich), anti-p62 (1:5000) (P0067; Sigma-Aldrich), and anti-14-3-3ε (1:800) (sc-23957; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-NSP2 antibodies were prepared by immunizing New Zealand white rabbits with a recombinant protein composed of the N-terminal 180 amino acids of NSP2 (NSP2-180). The monoclonal antibody against PRRSV-2 nucleocapsid (N) protein 6D10 was prepared in our laboratory [44 (link)]. Horseradish-peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies were purchased from Jackson Laboratories (1:5000) (West Grove, PA, USA).
Complete™ protease inhibitor cocktail (04693132001) was purchased from Roche (Basel, Switzerland), the aggresome detection kit (ab139486) was purchased from Abcam (Cambridge, UK), and nocodazole (M1404) and MG132 (C2211) were obtained from Sigma-Aldrich.

Cao S., Liu J., Ding G., Shao Q., Wang B., Li Y., Feng J., Zhao Y., Liu S, & Xiao Y. (2020). The tail domain of PRRSV NSP2 plays a key role in aggrephagy by interacting with 14-3-3ε. Veterinary Research, 51, 104.

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Immunoprecipitation of 14-3-3ε and PP1γ2 from Testis Extracts

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Testis extracts were pre-cleaned with protein G-sepharose 4 Fast Flow beads (GE Healthcare), incubated for 2 hours at 4°C and then washed three times with HB + by centrifugation at 8000 × g for 2 minutes. The beads were then incubated overnight at 4°C with gentle rocking with primary antibodies [goat polyclonal 14-3-3ε (R&D #AF4419); mouse monoclonal 14-3-3ε (Santa Cruz #SC23957) and rabbit PP1γ2 (YenZym) antibody]. Serum (goat, mouse, and rabbit serum) was used as a negative control. The beads were then washed two times with homogenizing buffer. The pre-cleared extracts/lysates were added to the beads/antibodies and incubated for four hours at 4°C, and then washed 5 times with TTBS and resuspended with 2XSDS reducing sample buffer boiled for 10 minutes. The beads were centrifuged at 10 000 × g for 10 minutes, and the supernatants were used for Western blot analysis.

Eisa A., Dey S., Ignatious A., Nofal W., Hess R.A., Kurokawa M., Kline D, & Vijayaraghavan S. (2020). The protein YWHAE (14-3-3 epsilon) in spermatozoa is essential for male fertility. Andrology, 9(1), 312-328.

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Immunoprecipitation and Protein Extraction

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Cells in the exponential growth phase were collected and washed. Ice-cold lysis buffer was added, and the cell suspension was sonicated. The supernatant was collected and the total protein concentration was determined. Samples (500 μg) were then incubated with 2 μg of YWHAE (1:100, sc-23957, Santa Cruz Biotechnology, Santa Cruz, CA) or HE4 (1:1500, ab200828, Abcam, Cambridge, UK) primary antibodies. An IgG antibody (5145S, Cell Signaling Technology Inc., Danvers, MA) of the same species as the primary antibodies was used as a negative control. Following mixing, the samples were rotated slowly overnight at 4 °C. Subsequently, Protein A/G Plus-Agarose Beads (sc-2003, Santa Cruz Biotechnology, Santa Cruz, CA) were added to each tube and incubated for 6 h. Bound proteins were collected via centrifugation, (2×) loading buffer was added, and the samples were boiled to denature the proteins.

Li X., Wang C., Wang S., Hu Y., Jin S., Liu O., Gou R., Nie X., Liu J, & Lin B. (2021). YWHAE as an HE4 interacting protein can influence the malignant behaviour of ovarian cancer by regulating the PI3K/AKT and MAPK pathways. Cancer Cell International, 21, 302.

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Dual-Labeling Immunofluorescence Assay

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Cells were seeded on a microscope slide and washed with phosphate-buffered saline (PBS) after they had adhered to the glass. Goat serum blocking solution was added, followed by the anti-YWHAE (1:100, sc-23957, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-HE4 (1:200, DF8160, Affinity Biosciences, Cincinnati, OH) primary antibody mix. A secondary antibody mixture containing tetramethylrhodamine-labelled goat anti-rabbit IgG (SA00007-2, Proteintech Group Inc., Wuhan, China) and fluorescein isothiocyanate-labelled goat anti-mouse IgG (SA00003-1, Proteintech Group Inc., Wuhan, China) was dropped onto the slide, which was then incubated for 2 h in the dark. Nuclei were stained with 4′,6-diamidino-2-phenylindole (4083S; Cell Signaling Technology Inc., Danvers, MA), and an anti-quenching agent was added dropwise onto the slide immediately before viewing the slices on a confocal microscope.

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Immunoprecipitation of YWHAE and HE4

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Cells in exponential growth were collected and washed. Iced lysis buffer was added and the cell suspension was sonicated. The supernatant was collected and total protein concentration was determined. Total protein samples (500 μg) were incubated with 2 μg of YWHAE (1 100, sc-23957, Santa Cruz Biotechnology, Santa Cruz, CA) or HE4 (1:1500, ab200828, Abcam, Cambridge, UK) primary antibody. An IgG antibody (5145S, Cell Signaling Technology, Beverly, MA) of the same species of the primary antibody was used as negative control. After mixing, the samples were left overnight at 4°C with slow rotation. Afterwards, beads (sc-2003, Santa Cruz Biotechnology, Santa Cruz, CA) were added to each tube and incubated for 6 h. The bound proteins were collected by centrifugation, (2×) Loading Buffer was added, and the samples were heated to denature the proteins.

Li X., Wang C., Wang S., Hu Y., Jin S., Liu O., Gou R., Nie X., Liu J., & Lin B. (2021). YWHAE Influences the Malignant Behaviour of Ovarian Cancer by Regulating the PI3K/AKT and MAPK Pathways Via HE4.

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Dual-Immunofluorescence Staining Protocol

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The cells were seeded on a microscope slide and washed with PBS after they adhered to the glass. Goat serum blocking solution was added, followed by the anti-YWHAE (1:100, sc-23957, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-HE4 (1:200, DF8160, A nity, OH, USA) primary antibody mix. A secondary antibody mixture containing tetramethylrhodamine-labelled goat anti-rabbit IgG (SA00007-2, Proteintech, Wuhan, China) and uorescein isothiocyanate-labelled goat anti-mouse IgG (SA00003-1, Proteintech, Wuhan, China) was dropped onto the slide, which was then incubated for 2 h in the dark. 4′,6diamidino-2-phenylindole (4083S; Cell Signaling Technology, Beverly, MA, USA) was used to stain the cell nucleus, and an anti-quenching agent was added dropwise onto the slide immediately before assessing the slices on a confocal microscope.

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