Ab3114

Manufactured by Abcam

Sourced in United Kingdom

Ab3114 is a primary antibody produced in rabbit. It is designed for use in Western blotting applications.

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Lab products found in correlation

Nanodrop, by Thermo Fisher Scientific (2 mentions) Sc 376865, by Santa Cruz Biotechnology (1 mentions) Immobilon forte western hrp substrate, by Merck Group (1 mentions) Ab126353, by Abcam (1 mentions) Ab33499, by Abcam (1 mentions) Ab14955, by Abcam (1 mentions) Ab1791, by Abcam (1 mentions) Sc 397, by Santa Cruz Biotechnology (1 mentions) Chk2 pt68, by Cell Signaling Technology (1 mentions) Novex nupage sds page gel system, by Thermo Fisher Scientific (1 mentions) Ms 285 p1, by Thermo Fisher Scientific (1 mentions) Chk1 ps345, by Cell Signaling Technology (1 mentions) Bradford assay, by Bio-Rad (1 mentions) Anti rad51 antibody, by Santa Cruz Biotechnology (1 mentions) Anti γh2ax, by Merck Group (1 mentions)

3 protocols using ab3114

Affinity Selection of Binding Proteins

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Affinity selection of binding proteins using a biotinylated 5–10–5 PS-MOE gapmer PS-ASO precoated on neutravidin beads was performed essentially as described (5 (link)). Beads bound proteins were then eluted by competition with 25 ul of 25 uM ASOs of the same sequence but different design, as shown in figure legend. Eluted proteins were separated on 4–12% SDS-PAGE, following by silver staining using silver staining kit (), based on the manufactorer's protocol. Additionally, proteins in PAGE gel were transferred to membrane, and detected by western alayses. The membranes were blocked with 5% non-fat dry milk in 1 × PBS at room temperature for 30 min. Membranes were then incubated with primary antibodies at room temperature for 2 h or at 4°C overnight. After three washes with 1× PBS, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:2000) at room temperature for 1 h to develop the image using Immobilon Forte Western HRP Substrate (Millipore). Primary antiobodies used for western analysis are: P54nrb (sc-376865, Santa Cruz Biotech.), PSF (Sc-271796, Santa Cruz Biotech.), RNase H1 (15606-1-AP, ProteinTech), Ku70 (ab3114, Abcam).

Vasquez G., Freestone G.C., Wan W.B., Low A., De Hoyos C.L., Yu J., Prakash T.P., Ǿstergaard M.E., Liang X.H., Crooke S.T., Swayze E.E., Migawa M.T, & Seth P.P. (2021). Site-specific incorporation of 5′-methyl DNA enhances the therapeutic profile of gapmer ASOs. Nucleic Acids Research, 49(4), 1828-1839.

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Protein Extraction and Western Blot Analysis

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Whole cell extracts were obtained by washing cells in phosphate-buffered saline (PBS) before lysis in SDS loading buffer (2% SDS, 10% (v/v) glycerol, 25 mM TCEP and 62.5 mM Tris-HCl, pH 6.8). Extracts were heated at 95 °C for 5 min, followed by shearing with 10 strokes through a 25G needle. Protein concentrations were determined by Bradford assay (Bio-Rad) or NanoDrop (Thermo Scientific). SDS-PAGE and western blotting were performed using the Novex NuPAGE SDS-PAGE gel system (Life Technologies) or the SE400 and TE42 systems from Hoefer. The following antibodies were used at the indicated dilutions: CHK1 (sc8408, Santa Cruz Biotechnology, 1/1000), CHK1-pS345 (2348, Cell Signaling Technology, 1/10,000), CHK2-pT68 (2661, Cell Signaling Technology, 1/500), DNA-PKcs (MS-369-P1, Thermo Scientific, 1/200), GFP (11814460001, Roche, 1/5000), γH2AX (05-636, Millipore, 1/1000), H3 (ab1791, Abcam, 1/50,000), H3-pS10 (ab14955, Abcam, 1/5000), Ku70 (ab3114, Abcam, 1/1000), Ku80 (MS-285-P1, Thermo Scientific, 1/2000), p21 (sc397, Santa Cruz Biotechnology, 1/1000), p53 (554293, BD Biosciences, 1/6000), PAXX (ab126353, Abcam, 1/500), RPA1, (A300-241A, Bethyl Laboratories, 1/1000), TopBP1 (A300-111A, Bethyl Laboratories, 1/5000), XLF (ab33499, Abcam, 1/650) and XRCC4 (ab145, Abcam, 1/3000).

Ochi T., Blackford A.N., Coates J., Jhujh S., Mehmood S., Tamura N., Travers J., Wu Q., Draviam V.M., Robinson C.V., Blundell T.L, & Jackson S.P. (2015). PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair. Science (New York, N.Y.), 347(6218), 185-188.

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Immunoblotting of DNA Repair Proteins

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Chemicals were from Sigma (Poole, UK) unless stated otherwise. Antibodies: anti-RAD51 antibody (rabbit polyclonal, sc-8349), from Santa Cruz Biotechnology, Santa Cruz, CA); Ku70 (monoclonal, ab3114), Ku80 (monoclonal, ab3107), from Abcam, Cambridge, UK; actin (mouse, monoclonal, Ab-1; Calbiochem, Merck Biosciences, Nottingham, UK); anti-γH2AX (monoclonal, 05-636; Millipore, Durham, UK).

Cornell L., Munck J., Alsinet C., Villanueva A., Ogle L., Willoughby C., Televantou D., Thomas H., Jackson J., Burt A., Newell D., Rose J., Manas D.M., Shapiro G., Curtin N, & Reeves H.L. (2014). DNA-PK – a candidate driver of hepatocarcinogenesis and tissue biomarker that predicts response to treatment and survival. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(4), 925-933.

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