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Zscan4

Manufactured by Merck Group
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ZSCAN4 is a laboratory equipment product designed for research purposes. It is a specialized tool used for the analysis and detection of specific genetic sequences. The core function of ZSCAN4 is to enable researchers to identify and study the ZSCAN4 gene, which plays a role in cellular processes. This product provides researchers with a reliable and efficient means of conducting genetic research and analysis.

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Lab products found in correlation

Nuclear extraction kit, by Active Motif (2 mentions) Supersignal west femto, by Thermo Fisher Scientific (2 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions) Accutase, by Merck Group (2 mentions) Zscan4, by OriGene (2 mentions) Zscan4, by Thermo Fisher Scientific (2 mentions) Lamin b, by Santa Cruz Biotechnology (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Ax15836, by Bio-Techne (2 mentions) Phospho p38 thr180 tyr182, by Cell Signaling Technology (2 mentions) Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (2 mentions) Cycloheximide (chx), by Merck Group (2 mentions) Flag tag (flag), by Merck Group (2 mentions) Erk1 2, by Santa Cruz Biotechnology (2 mentions) Mbd3l2, by Abcam (1 mentions) Oct3 4, by Santa Cruz Biotechnology (1 mentions) Ssea 4, by Merck Group (1 mentions) Ssea3, by Merck Group (1 mentions) Tra 1 60, by Merck Group (1 mentions) Anti h3k4me3, by Merck Group (1 mentions)

6 protocols using zscan4

1

Nuclear Protein Extraction and Analysis

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Nuclear proteins were fractionated using Nuclear Extraction Kit following manufacture's protocol (Active Motif). Total cell lysate was prepared in RIPA buffer and sonicated. For the detection of endogenous ZSCAN4 in Tu167 cells, cells were harvested by accutase (Millipore) and Cytoskeleton buffer (10 mM PIPES, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA and 0.5% Triton X100) was used to fractionate cytosolic proteins. Then, pellets were lysed in urea solution (8 M Urea in 0.01 Tris pH 8 + 0.1 M NaH2PO4) and sonicated. Nuclear proteins were electrophoresed in 8% polyacrylamide gels and transferred to a PVDF membrane. Immunoblot was performed using the following primary antibodies: ZSCAN4 (Origene; 1:1000), GAPDH (Santa Cruz; 1:5000), Actin (Sigma; 1:500), Lamin B (Santa Cruz; 1:2000) and with HRP (horseradish peroxidase) conjugated secondary antibodies (Millipore; 1:5000). Protein bands were detected using Pierce ECL Western Blotting Substrate (Thermo Scientific). SuperSignal West Femto (Thermo Scientific) was used to detect endogenous ZSCAN4 in Tu167 cells. All immunoblots shown represent at least 3 independent experiments.
Portney B.A., Khatri R., Meltzer W.A., Mariano J.M, & Zalzman M. (2018). ZSCAN4 is negatively regulated by the ubiquitin-proteasome system and the E3 ubiquitin ligase RNF20. Biochemical and biophysical research communications, 498(1), 72-78.
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2

Pluripotent Stem Cell Characterization

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For immunostaining, the primary antibodies used included those against OCT3/4 (1:500, Santa Cruz), SOX2 (1:500, Santa Cruz), NANOG (1:500, Abcam), SSEA3 (1:50, Millipore), SSEA4 (1:50, Millipore), TRA-1–60 (1:50, Millipore), UTF1 (1: 200, Abcam), DPPA3 (1:50, Santa Cruz), ZSCAN4 (1:100, Millipore) and MBD3L2 (1:100, Abcam). The following secondary antibodies were used: Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:500; Invitrogen), fluorescein isothiocyanate (FITC) 488-conjugated donkey anti-rabbit IgG (1:500; Invitrogen), and FITC 488-conjugated donkey anti-goat IgG (1:500; Invitrogen). Anti-H3K4me2 (Millipore), Anti-H3K4me3 (Millipore), Anti-H3K27me3 (Abcam) and Anti-H3K9me3 (Millipore) antibodies were used for ChIP experiments.
Wang Y., Zhao C., Hou Z., Yang Y., Bi Y., Wang H., Zhang Y, & Gao S. (2018). Unique molecular events during reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) at naïve state. eLife, 7, e29518.
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3

mESC Culture and Antibody Validation

Cited 1 time
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We used the mESC line E14Tg2a in this study. Cells were cultured on 0.1% gelatin-coated (Millipore) plates in DMEM (Life Technologies) supplemented with 15% fetal bovine serum (Sigma), penicillin/streptomycin, nonessential amino acid, sodium pyruvate, GlutaMax, β-mercaptoethanol (Life Technologies), and 1000 U/mL LIF (ESGRO, Millipore) unless specified. Antibodies used in this study were 5hmC (Active Motif, 39769), Oct4 (Santa Cruz Biotechnology, sc-8628), Nanog (Bethyl Laboratories, IHC-00205), Sox2 (Millipore, Ab5603), Tet1 (Ito et al. 2010 (link)), Tet2 (described below), Tet3 (Gu et al. 2011 (link)), Zscan4 (Millipore, Ab4340), Suz12 (Cao et al. 2002 (link)), Ring1b (Cell Signaling, 5694), Ezh2 (Cell Signaling, 5246), and Kap1 (Cell Signaling, 4123). Tet2 antibody was generated using HIS-tagged recombinant Tet2 2-374 expressed in Escherichia coli and purified using Talon superflow metal affinity resin (Clontech). Rabbit immunization was carried out by Pocono Rabbit Farm and Laboratory, Inc. The antiserum was affinity-purified using immunization antigen. The specificity of the affinity-purified antibody was confirmed using extracts from the control and Tet2 knockdown mESCs.
Lu F., Liu Y., Jiang L., Yamaguchi S, & Zhang Y. (2014). Role of Tet proteins in enhancer activity and telomere elongation. Genes & Development, 28(19), 2103-2119.
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4

Examining Stem Cell Regulatory Pathways

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Antibodies used were ERK5 (MRC-PPU Reagents & Services, Dundee), phospho-p38 (Thr180/Tyr182, Cell Signalling), phospho-ERK1/2 (Thr202/Tyr204, Cell Signalling), ERK1/2 (Santa Cruz Biotechnology), GFP (Abcam), KLF2 (Millipore), FLAG (Sigma), ZSCAN4 (Millipore), phospho-KLF2 (DSTT, Dundee), HA (Sigma). Inhibitors used were AX15836 (Tocris), XMD8-92 (N. Gray, Harvard), cycloheximide (Sigma).
Brown H.A., Williams C.A., Zhou H., Rios-Szwed D., Fernandez-Alonso R., Mansoor S., McMulkin L., Toth R., Gourlay R., Peltier J., Dieguez-Martinez N., Trost M., Lizcano J.M., Stavridis M.P, & Findlay G.M. (2021). An ERK5–KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells. Biochemical Journal, 478(23), 4119-4136.
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5

Nuclear Protein Extraction and Analysis

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Nuclear proteins were fractionated using Nuclear Extraction Kit following manufacture's protocol (Active Motif). Total cell lysate was prepared in RIPA buffer and sonicated. For the detection of endogenous ZSCAN4 in Tu167 cells, cells were harvested by accutase (Millipore) and Cytoskeleton buffer (10 mM PIPES, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA and 0.5% Triton X100) was used to fractionate cytosolic proteins. Then, pellets were lysed in urea solution (8 M Urea in 0.01 Tris pH 8 + 0.1 M NaH2PO4) and sonicated. Nuclear proteins were electrophoresed in 8% polyacrylamide gels and transferred to a PVDF membrane. Immunoblot was performed using the following primary antibodies: ZSCAN4 (Origene; 1:1000), GAPDH (Santa Cruz; 1:5000), Actin (Sigma; 1:500), Lamin B (Santa Cruz; 1:2000) and with HRP (horseradish peroxidase) conjugated secondary antibodies (Millipore; 1:5000). Protein bands were detected using Pierce ECL Western Blotting Substrate (Thermo Scientific). SuperSignal West Femto (Thermo Scientific) was used to detect endogenous ZSCAN4 in Tu167 cells. All immunoblots shown represent at least 3 independent experiments.
Portney B.A., Khatri R., Meltzer W.A., Mariano J.M, & Zalzman M. (2018). ZSCAN4 is negatively regulated by the ubiquitin-proteasome system and the E3 ubiquitin ligase RNF20. Biochemical and biophysical research communications, 498(1), 72-78.
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6

Molecular Analysis of mESC Signaling

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Antibodies used were ERK5 (MRC-PPU Reagents & Services, Dundee), phospho-p38 (Thr180/Tyr182, Cell Signalling), phospho-ERK1/2 (Thr202/Tyr204, Cell Signalling), ERK1/2 (Santa Cruz Biotechnology), GFP (Abcam), KLF2 (Millipore), FLAG (Sigma), ZSCAN4 (Millipore), phospho-KLF2 (DSTT, Dundee), HA (Sigma). Inhibitors used were AX15836 (Tocris), XMD8-92 (N. Gray, Harvard), cycloheximide (Sigma). mESC culture, transfection and lysis CCE mESCs were cultured on gelatin coated plates in media containing LIF, 10% fetal calf serum (Gibco), and 5% knockout serum replacement (Invitrogen) unless otherwise stated. mESCs were transfected with pCAGGS expression vectors using Lipofectamine LTX (Life Technologies), selected with puromycin after 24 h and cultured for the stated times. For CRISPR/Cas9, mESCs were transfected with pX335 and pKN7 (Addgene) and selected, then either lysed or clones isolated. Cell extracts were made in lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% NP-40 [v/v], 0.5% sodium deoxycholate [w/v], 10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM NaF, 2 mM Na3VO4, and Roche Complete Protease Inhibitor Cocktail Tablets).
Brown H.A., Williams C.A.C., Zhou H., Rios-Szwed D., Fernández-Alonso R., Mansoor S., McMulkin L., Toth R., Gourlay R., Peltier J., Diéguez-Martínez N., Trost M., Lizcano J.M., Stavridis M.P., & Findlay G.M. (2021). An ERK5-KLF2 signalling module regulates early embryonic gene expression dynamics and stem cell rejuvenation.
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