Clone 13e5

Manufactured by Cell Signaling Technology

Sourced in United States

The Clone 13E5 is a mouse monoclonal antibody that recognizes a specific antigen within the cell. It can be used in various laboratory techniques, including immunohistochemistry, Western blotting, and flow cytometry, to detect and study the target protein. The core function of this product is to provide a specific and reliable tool for researchers to investigate cellular processes and pathways.

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Lab products found in correlation

Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions) Wet tank blotting system, by Bio-Rad (2 mentions) Dnmt1, by Abcam (2 mentions) Nbp2 20171, by Novus Biologicals (2 mentions) Clone 8h3, by Cell Signaling Technology (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Protease inhibitor, by Solarbio (1 mentions) Chemiluminescent hrp substrate kit, by Merck Group (1 mentions) Chemidoc xrs system, by Bio-Rad (1 mentions) Ack lysing buffer, by Thermo Fisher Scientific (1 mentions) Ecl reagent, by Bio-Rad (1 mentions) Protein assay, by Bio-Rad (1 mentions) Alexa fluor 647 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions) Rad23a, by Cell Signaling Technology (1 mentions) Clone da1e, by Cell Signaling Technology (1 mentions) Clone 1b1b2, by Cell Signaling Technology (1 mentions) Clone m2, by Merck Group (1 mentions) Clone m01, by Abnova (1 mentions)

Close spelling variants of this product

Anti-βactin antibody (clone 13E5, Rabbit anti-β-Actin clone 13E5 β-actin (clone 13E5) Anti-β-actin (clone 13E5,

9 protocols using clone 13e5

Western Blotting of DNA Repair Proteins

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Proteins isolated from cells with NP-40 lysis buffer were resolved using NuPAGE 4–12% Bis-Tris gel (ThermoFisher) and transferred from gel to PVDF membrane using Wet/Tank Blotting Systems (Bio-Rad). Membrane was blocked with 5% non-fat milk in TBSTE buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 0.05% Tween-20, 1mM EDTA), incubated with indicated primary antibodies, followed by secondary antibodies conjugated with horse-radish peroxidase (HRP) and signal was detected with enhanced chemiluminescence reagents (Invitrogen) and X-ray film. Antibodies against ATRX (1:1000, clone D5), FANCD2 (1:1000, clone Fl17) and BLM (1:1000, clone B4) were purchased from Santa Cruz Biotechnology. Antibodies against RNASE H1 (1:1000, NBP2–20171) from Novus biologicals, and WRN (1:1000, clone 8H3) and β-ACTIN HRP (1:5000, clone 13E5) from Cell signaling and DNMT1 (1:1000, ab19905) from Abcam.

Shukla V., Samaniego-Castruita D., Dong Z., González-Avalos E., Yan Q., Sarma K, & Rao A. (2021). TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures. Nature immunology, 23(1), 99-108.

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Protein Expression Analysis in Transgenic Mice

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Tissue lysates were obtained from 1 TG per mouse collected in 200 μl RIPA buffer (PIERCE, Thermo Scientific) with protease inhibitor cocktail (Roche) and disrupted by sonication on ice. Thirteen microliters of tissue lysates were loaded onto 4-12% NuPAGE gel, transfer onto nitrocellulose membrane and probe for m4 expression using rabbit anti-HA antibody (1:1000 mAb clone C29F, Cell signaling) and β-actin (1: 1000 mAb clone13E5, cell signaling) for protein loading. Membrane hybridization and detection were performed using PIERCE Fast Western blotting kit Super signal, West pico Rabbit (Thermo Scientific) per manufacturer protocol and imaged using ChemiDoc Imaging system (BIO-RAD).

Aubert M., Haick A.K., Strongin D.E., Klouser L.M., Loprieno M.A., Stensland L., Santo T.K., Huang M.L., Hyrien O., Stone D, & Jerome K.R. (2024). Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo. Nature Communications, 15, 4018.

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Quantification of DROSHA and DICER knockdown

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Jurkat cells were cultured in RPMI + 10% FCS. They were transduced with lentiviral shRNAs against DROSHA, DICER or an empty vector. After 3 days, the transduced cells (GFP⁺) were sorted to purity and returned to culture for an additional 3 days to expand. The cells were then harvested, washed in cold PBS and lysed in RIPA buffer. The lysates were then analysed for DROSHA or DICER protein expression by Western blotting. Analysis of β‐actin employed as a loading control. Separate gels were run to detect β‐actin due to the difference in detection sensitivity compared with the detection of the RNase III enzymes. Lysates from 5 × 10⁶ cells were loaded for the DROSHA and DICER blots, while lysates from 2 × 10⁵ cells were loaded for the β‐actin blots. The rabbit monoclonal antibodies to detect DROSHA (clone EPR12794) and DICER (clone EPR24104‐105) were purchased from Abcam (Cambridge), while the rabbit monoclonal antibody to detect β‐actin (clone 13E5) was purchased from Cell Signaling Technology (Danvers).

Gu K., Walpole C., Gooneratne S., Liu X., Haigh O.L., Radford K.J, & Chong M.M. (2022). DROSHA but not DICER is required for human haematopoietic stem cell function. Clinical & Translational Immunology, 11(1), e1361.

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Western Blotting of DNA Repair Proteins

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Quantifying NY-ESO-1 and DNMT3a Protein Levels

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Cells and tumor tissues were lysed in RIPA buffer containing protease inhibitors (Solarbio, Beijing, China). Proteins were size-fractionated on 10–12% PAGE gels and transferred to Immobilon-transfer nitrocellulose membranes (Millipore, Burlington, MA, USA). The membranes were incubated with anti-human NY-ESO-1 (1:250 dilution; Clone SP349; Abcam, Cambridge, MA, USA), anti-DNMT3 (1:2000; Clone EPR18455, Abcam), and anti-β-actin (1:2000 dilution; Clone 13E5; Cell Signaling Technology, Danvers, MA, USA), washed, and incubated with an HRP-labeled secondary antibody. Protein bands were detected with the Chemiluminescent HRP Substrate Kit (Millipore). The densitometry readings/intensity ratios were analyzed with the ChemiDoc XRS + System (Bio-Rad, Hercules, CA, USA) by comparing the protein band intensity of NY-ESO-1 or DNMT3a to the protein band intensity of β-actin.

Kang S., Wang L., Xu L., Wang R., Kang Q., Gao X, & Yu L. (2022). Decitabine enhances targeting of AML cells by NY-ESO-1-specific TCR-T cells and promotes the maintenance of effector function and the memory phenotype. Oncogene, 41(42), 4696-4708.

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Analyzing Myd88 Expression in Murine Organs

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Lung, liver, kidney, and spleens were harvested from NOD.B10^Myd88Δ, NOD.B10^Myd88fl/fl, NOD.B10^{LSL−/−Vav+}, NOD.B10^LSL−/− and NOD.B10^LSL+/− females. For western blotting experiments, lung, liver and kidney were placed in RIPA buffer and snap frozen. Tissue was homogenized by sonication. Spleens were mechanically dissociated, RBCs removed (ACK lysing buffer (Gibco)), and 1.0 × 10⁷ cells were lysed in RIPA buffer. Protein was quantified by BioRad protein assay (BioRad Laboratories). All samples were solubilized using Laemmli buffer, separated by electrophoresis and transferred to nitrocellulose membranes. Membranes were blotted overnight at 4°C with antibodies directed against Myd88 (Cell Signaling Technology, clone D80F5) and β-actin (Cell Signaling Technology, clone 13E5). Membranes were incubated with HRP-conjugated secondary antibodies at RT for 1 hr and developed using ECL reagents (BioRad Laboratories). For qPCR analyses, lung, liver, kidney, and spleen were snap frozen. After mRNA isolation, cDNA synthesis was performed and qPCR carried out as previously described using SYBR green [10 (link)]. Primer sequences employed were as follows: Myd88: Forward: 5’-AGAGCTGCTGGCCTTGTTAG-3’ Reverse: 5”-CGAAAAGTTCCGGCGTTTGT-3’ and β-Actin: Forward: 5’-TGTTACCAACTGGGACGACA-3’, Reverse: 5’-GGGGTGTTGAAGGTCTCAAA -3’.

Kiripolsky J., Kasperek E.M., Zhu C., Li Q.Z., Wang J., Yu G, & Kramer J.M. (2021). Tissue-specific activation of Myd88-dependent pathways governs disease severity in primary Sjögren’s syndrome. Journal of autoimmunity, 118, 102608.

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Immunoprecipitation and Immunoblotting Protocol

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Immunoprecipitation and immunoblotting experiments were performed as previously described.^{34 (link)} Antibodies against HA (1:2000; clone C29F4, cat # 3724S), β-actin (1:5000; clone 13E5, cat # 4970S), H3 (1:4000; clone 1B1B2, cat # 14269S) and RAD23A (1:1000; clone D7U7Z, cat # 24555) were purchased from Cell Signaling Technology, as was a non-specific isotype control IgG (clone DA1E, cat # 3900S). The FLAG antibody was from Sigma-Aldrich (1:4000; clone M2, cat # F1804). The Pol ι antibody was from Abnova (1:1000; clone M01, cat # H00011201-M01). Primary antibodies were detected with fluorescent secondary antibodies, and immunoblots quantified, as described previously.^{34 (link)} For the microarray, the Pol ι antibody was detected with an Alexa Fluor 647-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific cat #A-21240).

Ashton N.W., Jaiswal N., Moreno N.C., Semenova I.V., D’Orlando D.A., Latancia M.T., McIntyre J., Woodgate R, & Bezsonova I. (2023). A Novel Interaction Between RAD23A/B and Y-family DNA Polymerases. Journal of molecular biology, 435(24), 168353.

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Quantifying Autophagy Markers in Tissues

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For detection of autophagy biomarkers in vivo, tissues were snap frozen, and then mechanically disrupted before cell lysis. For ATG7 detection, cell lines were subjected to standard lysis procedures. In both cases, 50 µg proteins were separated on NuPAGE® Novex® Bis-Tris 4–12% pre-cast gels (Invitrogen) and electrotransferred to polyvinyldifluoride (PVDF) membranes (Millipore Sigma). Membranes were blocked with 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk for 1 h and incubated overnight with primary antibody specific for MAP1LC3B (1:1000, #2775 from Cell Signaling Technology), SQSTM1 (1:1000, #5114 from Cell Signaling Technology), ATG7 (1:1000, clone D12B11, #8558 from Cell Signaling Technology; or 1:3000, clone ATG7-13, #SAB4200304 from Millipore Sigma), or ACTB (1:1000, clone 13E5, #4970 from Cell Signaling Technology; or 1:2000, clone 8H10D10, #3700 from Cell Signaling Technology), at 4 °C. Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated anti-mouse (#NA931, from GE Healthcare Life Sciences, 1:5000) or anti-rabbit (#NA934, from GE Healthcare Life Sciences, 1:5000) secondary antibodies and revealed with the Pierce™ ECL Plus chemiluminescent substrate (#32132, Thermo Fisher) on a C600 Gel Doc & Western Imaging System operated by cSeries Capture v. 1.6.8.1110 (Azure Biosystems).

Buqué A., Bloy N., Perez-Lanzón M., Iribarren K., Humeau J., Pol J.G., Levesque S., Mondragon L., Yamazaki T., Sato A., Aranda F., Durand S., Boissonnas A., Fucikova J., Senovilla L., Enot D., Hensler M., Kremer M., Stoll G., Hu Y., Massa C., Formenti S.C., Seliger B., Elemento O., Spisek R., André F., Zitvogel L., Delaloge S., Kroemer G, & Galluzzi L. (2020). Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer. Nature Communications, 11, 3819.

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Antibody Detection of TSPYL1, HDAC1, and Golgi

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The following antibodies were used: mouse polyclonal against human TSPYL1 (MaxPab H00007259-B01P; Abnova used at 1:1000 for immunoblot and 1:50 for immunostaining), rabbit polyclonal against human HDAC1 (Clone H-51; Santa Cruz used at 1:1000 for immunoblot), rabbit monoclonal human beta-actin (Clone 13E5, Cell Signaling Technology used at 1:1000 for immunoblot) and rabbit polyclonal golgi marker Mannosidase II (Abcam used at 1:200 for immunostaining).

Buyse G., Di Michele M., Wijgaerts A., Louwette S., Wittevrongel C., Thys C., Downes K., Ceulemans B., Van Esch H., Van Geet C, & Freson K. (2020). Unravelling the disease mechanism for TSPYL1 deficiency. Human molecular genetics, 29(20).

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