Psang10 3f bg4

Manufactured by Addgene

The PSANG10-3F-BG4 is a lab equipment product. It serves a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.

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Lab products found in correlation

Ni nta beads 6ff, by Smart-Lifesciences (1 mentions) Pdest frt t0 gfp brca1, by Addgene (1 mentions) Hoechst dye, by Santa Cruz Biotechnology (1 mentions) Scrambled sirna, by Qiagen (1 mentions) Prl tk renilla, by Promega (1 mentions) Mouse anti lamin b, by Santa Cruz Biotechnology (1 mentions) Flag agarose bead, by Merck Group (1 mentions) Bioruptor u200, by Thermo Fisher Scientific (1 mentions)

4 protocols using psang10 3f bg4

Purification of BG4-EGFP Antibody

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The scFv antibody, BG4, was purified using the expression vector pSANG10-3F-BG4 (Addgene plasmid no. 55756) according to the previous study (Hänsel-Hertsch et al. 2018 (link)). To get BG4-EGFP antibody, the EGFP coding sequence was amplified from the EGFP-Tpr (Addgene #35024) and subcloned into pSANG10-3F-BG4 to generate the BG4-EGFP expression plasmid, which was chemically transformed into BL21 (DE3) competent cells. Expression of BG4-EGFP was induced by 0.2 mM IPTG for 14 h at 16°C, 200 rpm. BG4-EGFP protein was then purified with the Ni NTA beads 6FF (Smart-Lifesciences). See Supplemental Methods for further details.

Li C., Wang H., Yin Z., Fang P., Xiao R., Xiang Y., Wang W., Li Q., Huang B., Huang J, & Liang K. (2021). Ligand-induced native G-quadruplex stabilization impairs transcription initiation. Genome Research, 31(9), 1546-1560.

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Molecular Tools for DNA Damage Response

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Pyridostatin (PDS) was obtained from Cayman Chemical (#18013). TmPyP4 was from Calbiochem (#613560). Hoechst dye was from Santa Cruz Biotechnology (#sc-394039). Antibodies against synapsin were from Synaptic Systems (#106002). Antibodies against BRCA1 (#ab191042) and 53BP1 (#ab172580) were from Abcam. Antibodies against γH2A.X (JBW301) were from EMD Millipore (#05636). Rabbit antibodies against MAP2c (H-300, #sc-20172), and mouse antibodies against MAP2c (A-4, #sc-74421) were from Santa Cruz Biotechnology. Antibodies against β-actin (8H10D10) were from Cell Signaling (#3700). Anti-rabbit Alexa Fluor 488–labeled (#A11008), anti-mouse Alexa Fluor 488–labeled (#A11001), anti-rabbit Alexa Fluor 546–labeled (A11010), and anti-mouse Alexa Fluor 546-labeled (#A11003) secondary antibodies were from Life Technologies. pCAG-mApple-hTP53BP1 (1220-1709aa) was synthesized by Vectorbuilder. pSANG10-3F-BG4 was from Addgene (#55756; deposited by Dr. Shankar Balasubramanian, the University of Cambridge). pDEST-FRT/T0-GFP-BRCA1 was from Addgene (#71116; deposited by Dr. Durocher, the University of Toronto).

Moruno-Manchon J.F., Koellhoffer E.C., Gopakumar J., Hambarde S., Kim N., McCullough L.D, & Tsvetkov A.S. (2017). The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons. Aging (Albany NY), 9(9), 1957-1970.

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Topoisomerase Depletion and Inhibition

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The del4 wildtype (#16604) and the pSang10-3F BG4 (#55756) plasmids were purchased from addgene, and the pRL-TK Renilla (#E2241) plasmid was purchased from Promega. The yeast S. cerevisiae strain RS190 lacking the endogenous TOP1 gene was a kind gift from R. Sternglanz (State University of New York, Stony Brook, NY). The plasmid pHT143, used for expression of TOP1, was described previously (40 (link)).
Rabbit anti-TOP1 (#A302-590A) and rabbit anti-TOP2α (#A300-054A) were purchased from Bethyl, mouse anti-TOP2β (#611493) from BD transduction, mouse anti-TBP (#ab818) from Abcam, and mouse anti-Lamin B (#sc-365214) were from Santa Cruz biotechnologies. All secondary antibodies were purchased from DAKO. siRNA targeting TOP1 (#3510923), TOP2α (#342786) and TOP2β (#3389257) as well as scrambled siRNA (#1027281) were from Qiagen, and the G4 drug PhenDC3 was from Sigma-Aldrich ApS.

Keller J.G., Hymøller K.M., Thorsager M.E., Hansen N.Y., Erlandsen J.U., Tesauro C., Simonsen A.K., Andersen A.B., Vandsø Petersen K., Holm L.L., Stougaard M., Andresen B.S., Kristensen P., Frøhlich R, & Knudsen B.R. (2022). Topoisomerase 1 inhibits MYC promoter activity by inducing G-quadruplex formation. Nucleic Acids Research, 50(11), 6332-6342.

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Purification of G4 DNA-binding Proteins

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pSANG10-3F-BG4 (Addgene 55756)52 (link) was transformed into BL21(DE3) competent Escherichia coli, (Invitrogen) grown in LB broth and induced for 6 h with 10 mM isopropyl-β-D-thiogalactoside (Life Technologies). Bacterial pellets were snap-frozen, lysed and sonicated (Fisher BioRuptor U200) before centrifuging at 12 K r.p.m. Lysates were incubated at 4 °C with the end-over-end rotation overnight with Flag-agarose beads (Sigma Aldrich). Beads were washed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% NP-40) and used immediately for IP go G4 DNA.

Day T.A., Layer J.V., Cleary J.P., Guha S., Stevenson K.E., Tivey T., Kim S., Schinzel A.C., Izzo F., Doench J., Root D.E., Hahn W.C., Price B.D, & Weinstock D.M. (2017). PARP3 is a promoter of chromosomal rearrangements and limits G4 DNA. Nature Communications, 8, 15110.

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