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Ncl l p53 cm5p

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The NCL-L-p53-CM5p is a laboratory equipment product from Leica. It is used for the detection of p53 protein in various biological samples. The product provides a specific and reliable tool for researchers and scientists in their investigations.

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Lab products found in correlation

Streptavidin hrp, by Thermo Fisher Scientific (2 mentions) Tyramide alexa fluor 568, by Thermo Fisher Scientific (2 mentions) Ez cytofunnel, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Anti n cadherin, by BD (2 mentions) Tsa kit 24, by Thermo Fisher Scientific (2 mentions) Vectastain abc hrp kit, by Vector Laboratories (2 mentions) Anti cardiac troponin t ct3 clone, by Developmental Studies Hybridoma Bank (2 mentions) Anti myosin heavy chain antibody mf20 clone, by Developmental Studies Hybridoma Bank (2 mentions) H3k4me3 antibody, by Abcam (2 mentions) Ab3623, by Merck Group (1 mentions) Anti mouse igg hrp, by GE Healthcare (1 mentions) Acetyl p53 lys379, by Cell Signaling Technology (1 mentions) Ab1791, by Abcam (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Ab1191, by Abcam (1 mentions) Ab4441, by Abcam (1 mentions) Ab18192, by Abcam (1 mentions) Anti rabbit igg hrp, by Bio-Rad (1 mentions) Spike in antibody, by Active Motif (1 mentions)

Spelling variants of this product

NCL-p53-CM5p (12 citations) P53-CM5P-L (2 citations)

10 protocols using ncl l p53 cm5p

1

Immunohistochemical Staining Protocol

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5-µm sections were rehydrated through ethanol series. Antigen retrieval was performed in 10 mM sodium citrate, pH 6, using a microwave. Antigen retrieval was performed in 10 mM sodium citrate pH6 using a microwave. Sections were then treated with 3% H2O2 to remove endogenous peroxidase activities. Sections were blocked in 10% goat serum, followed by avidin and biotin. Sections were then incubated in primary antibody (1:500) overnight at 4°C (p53, NCL-L-p53-CM5p, Leica Biosystems; cleaved caspase-3, MilliporeSigma AB3623). Sections were then washed thoroughly and incubated in biotinylated secondary antibody for 1 h at room temperature (secondary antibody, 1:750), followed by avidin-biotin complex for 30 min, and developed in DAB. Sections were counterstained in hematoxylin and dehydrated through an ethanol series.
Loe A.K., Francis R., Seo J., Du L., Wang Y., Kim J.E., Hakim S.W., Kim J.E., He H.H., Guo H, & Kim T.H. (2021). Uncovering the dosage-dependent roles of Arid1a in gastric tumorigenesis for combinatorial drug therapy. The Journal of Experimental Medicine, 218(6), e20200219.
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2

Immunostaining of Embryoid Bodies

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Day 9 EBs were harvested and fixed with 4% paraformaldehyde at 4°C overnight. EBs were sent to ULAM (Unit for laboratory animal medicine) at University of Michigan for paraffin processing, embedding, and sectioning. Antigen unmasking on slides was using citric acid methods as prescribed previously (Gage and Camper, 1997 (link)). Immunostaining was performed by combining of VectaStain ABC-HRP kit (Vector laboratories) and TSA Kit #24, with HRP-Streptavidin and Alexa Fluor 568 Tyramide (ThermoFisher Scientific). The primary antibodies were used for staining: anti-N-cadherin (mouse/monoclonal/1:2000, BD), anti- cardiac troponin T (CT3 clone, mouse/monoclonal/1:100, Developmental Studies Hybridoma Bank) and anti-myosin heavy chain antibody (MF20 clone, mouse/monoclonal/1:100, Developmental Studies Hybridoma Bank). For cytospin, Day 2 WT and Wdr5KO EBs were harvested and trypsinized to single cell suspension. Cells were attached to EZ cytofunnels (ThermoFisher) slides using Cytospin 3 (Shandon). Cells were fixed with 100% methanol and immunostaining was performed using p53 antibody (NCL-L-P53-CM5P, Leica biosystems) at 1:100 dilution. Counter nuclear staining was performed with DAPI (Molecular Probes). Control sections were incubated without primary antibodies. 4 representative pictures from different field were recorded under fluorescence microscope (Olympus DP73).
Li Q., Mao F., Zhou B., Huang Y., Zou Z., denDekker A.D., Xu J., Hou S., Liu J., Dou Y, & Rao R.C. (2020). p53 Integrates Temporal WDR5 Inputs during Neuroectoderm and Mesoderm Differentiation of Mouse Embryonic Stem Cells. Cell reports, 30(2), 465-480.e6.
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3

Chromatin Modification Antibody Validation

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H3K27ac (39,133, Active Motif Lot # 31814008), H3K27ac (39,685, Active Motif, no. 14517014), H3K18ac (ab1191, Abcam, no. GR3211480–1), H3K27me3 (9756, Cell Signaling), H3K9ac (ab4441, Abcam), H4K16ac (39,167, Active Motif), H3 general (ab1791, Abcam, no. GR177884–2), Spike-In antibody (61,686, Active Motif, Lot# 00419007), p300 (sc-584, Santa Cruz, Lot # F3016), Gapdh (2118, Cell Signaling, Lot # 10), CBP(D6C5) (7389S, Cell Signaling), p53(CM5) (NCL-L-p53-CM5p, Leica Biosystems), PKCs p2056 (ab18192, Abcam), KAP1/TRIM29 pS824 (A300-767A, Bethyl), Acetyl-p53 (Lys379) (2570, Cell Signaling), anti-mouse IgG-HRP (NA93V, GE, no.9773218), anti-rabbit IgG-HRP (170–6515, Bio-Rad, no. 350003248).
Martire S., Nguyen J., Sundaresan A, & Banaszynski L.A. (2020). Differential contribution of p300 and CBP to regulatory element acetylation in mESCs. BMC Molecular and Cell Biology, 21, 55.
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4

Western Blot Analysis of Metabolic Proteins

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Cell lysates were extracted using RIPA buffer and protein concentration was determined by BCA assay. Samples were boiled for 5 minutes and 20 to 30 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked with 3% milk prepared in 1X TBS-Tween and probed with the relevant primary antibody overnight at 4°C. Membranes were then incubated with horseradish peroxide (HRP)-conjugated secondary antibodies at room temperature and proteins were detected using Pierce ECL Western Blotting Substrate (34095, Thermo Fisher Scientific). Blots were imaged using HyBlot CL Autoradiography Film (E3018, Denville Scientific) and Konica Medical Film Processor (Model SRX-101A). Antibodies were diluted as follows: p53 (CM5) (1:500, NCL-L-p53-CM5p, Leica Biosystems), p21 (F-5) (1:500, sc-6246, Santa Cruz Biotechnology), p19 (M-167) (1:500, sc-1063, Santa Cruz Biotechnology), Ogdh (1:1000, 15212–1-AP, ProteinTech), Idh1 (1:1000, 12332–1-AP, ProteinTech), Sdha (1:1000, ab14715, Abcam) and Tubulin (1:10,000, T9026, Sigma-Aldrich).
Morris JP I.V., Yashinskie J.J., Koche R., Chandwani R., Tian S., Chen C.C., Baslan T., Marinkovic Z.S., Sánchez-Rivera F.J., Leach S.D., Carmona-Fontaine C., Thompson C.B., Finley L.W, & Lowe S.W. (2019). Alpha-ketoglutarate links p53 to cell fate during tumor suppression. Nature, 573(7775), 595-599.
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5

Cut & Run Assay for Chromatin Profiling

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Day 2 WT, Wdr5KO and DKO EBs were harvested and trypsinized to single cell suspension. 2×106 cells were prepared for Cut & Run experiment per antibody as previously described (Skene and Henikoff, 2017 (link); Skene et al., 2018 (link)) and the following antibodies were used: WDR5 antibody (Bethyl), P53 antibody (NCL-L-P53-CM5P, Leica biosystems) and H3K4me3 antibody (Abcam). Protein A–micrococcal nuclease (pA-MN) fusion protein were generously provided by Dr. Steven Henikoff.
Li Q., Mao F., Zhou B., Huang Y., Zou Z., denDekker A.D., Xu J., Hou S., Liu J., Dou Y, & Rao R.C. (2020). p53 Integrates Temporal WDR5 Inputs during Neuroectoderm and Mesoderm Differentiation of Mouse Embryonic Stem Cells. Cell reports, 30(2), 465-480.e6.
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6

Chromatin Immunoprecipitation of p53

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MEFs were grown in DMEM containing 10% FCS and seeded at 7 × 106 cells per 10 cm dish one day prior to the ChIP experiment. After treatment with 0.2 μg/ml doxorubicin for 6h, cells were harvested to prepare chromatin for immunoprecipitation using p53 polyclonal antibodies (NCL-L-p53-CM5p; Leica Biosystems). ChIPs were performed essentially as described previously (Kenzelmann Broz et al., 2013 (link)). Chromatin-immunoprecipitated DNA was quantified by qPCR using SYBR Green and a 7900HT Fast Real-Time PCR machine (Applied Biosystems) and primers specific for Eif4ebp1 (Table S2). The signals obtained from the ChIP were analyzed by the percent input method.
Tiu G.C., Kerr C.H., Forester C.M., Krishnarao P.S., Rosenblatt H.D., Raj N., Lantz T.C., Zhulyn O., Bowen M.E., Shokat L., Attardi L.D., Ruggero D, & Barna M. (2021). A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies. Developmental cell, 56(14), 2089-2102.e11.
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7

Immunostaining of Embryoid Bodies

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Day 9 EBs were harvested and fixed with 4% paraformaldehyde at 4°C overnight. EBs were sent to ULAM (Unit for laboratory animal medicine) at University of Michigan for paraffin processing, embedding, and sectioning. Antigen unmasking on slides was using citric acid methods as prescribed previously (Gage and Camper, 1997 (link)). Immunostaining was performed by combining of VectaStain ABC-HRP kit (Vector laboratories) and TSA Kit #24, with HRP-Streptavidin and Alexa Fluor 568 Tyramide (ThermoFisher Scientific). The primary antibodies were used for staining: anti-N-cadherin (mouse/monoclonal/1:2000, BD), anti- cardiac troponin T (CT3 clone, mouse/monoclonal/1:100, Developmental Studies Hybridoma Bank) and anti-myosin heavy chain antibody (MF20 clone, mouse/monoclonal/1:100, Developmental Studies Hybridoma Bank). For cytospin, Day 2 WT and Wdr5KO EBs were harvested and trypsinized to single cell suspension. Cells were attached to EZ cytofunnels (ThermoFisher) slides using Cytospin 3 (Shandon). Cells were fixed with 100% methanol and immunostaining was performed using p53 antibody (NCL-L-P53-CM5P, Leica biosystems) at 1:100 dilution. Counter nuclear staining was performed with DAPI (Molecular Probes). Control sections were incubated without primary antibodies. 4 representative pictures from different field were recorded under fluorescence microscope (Olympus DP73).
Li Q., Mao F., Zhou B., Huang Y., Zou Z., denDekker A.D., Xu J., Hou S., Liu J., Dou Y, & Rao R.C. (2020). p53 Integrates Temporal WDR5 Inputs during Neuroectoderm and Mesoderm Differentiation of Mouse Embryonic Stem Cells. Cell reports, 30(2), 465-480.e6.
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8

Cut & Run Assay for Chromatin Profiling

Cited 1 time
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Day 2 WT, Wdr5KO and DKO EBs were harvested and trypsinized to single cell suspension. 2×106 cells were prepared for Cut & Run experiment per antibody as previously described (Skene and Henikoff, 2017 (link); Skene et al., 2018 (link)) and the following antibodies were used: WDR5 antibody (Bethyl), P53 antibody (NCL-L-P53-CM5P, Leica biosystems) and H3K4me3 antibody (Abcam). Protein A–micrococcal nuclease (pA-MN) fusion protein were generously provided by Dr. Steven Henikoff.
Li Q., Mao F., Zhou B., Huang Y., Zou Z., denDekker A.D., Xu J., Hou S., Liu J., Dou Y, & Rao R.C. (2020). p53 Integrates Temporal WDR5 Inputs during Neuroectoderm and Mesoderm Differentiation of Mouse Embryonic Stem Cells. Cell reports, 30(2), 465-480.e6.
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9

Multiparametric Immune Cell Profiling

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CD45 (103138, clone 30-F11), CD3 (100206, clone 17A2), CD4 (100411, clone GK1.5), CD8 (100714, clone 53.6.7), CD19 (115520, clone 6D5), NKp46 (137610, clone 29A1.4), CD11b (101212, clone M1/70), Ly6G (127624, clone 1A8), CD11c (117318, clone N418), F4/80 (123128, clone BM8) were purchased from Biolegend for flow cytometry. SRSF1 (ab129108; Abcam), GFP (2956T; Cell Signaling Technology), CK19 (TROMA III, DSHB), phospho-ERK1/2 (4370, Cell Signaling), ERK1/2 (4695, Cell Signaling), GFP (2956, Cell Signaling), mIL1R1 (AF771-SP, Novus Biologicals), hIL1R1 (ab106278; Abcam), CD45 (70257, Cell Signaling), Cleaved Caspase-3 (9661, Cell Signaling), F4/80 (70076, Cell Signaling), P53 (NCL-L-p53-CM5p, Leica Biosystems) were used as primary antibodies for western blotting, immunohistochemistry, and immunofluorescence.
Wan L., Lin K.T., Rahman M.A., Ishigami Y., Wang Z., Jensen M.A., Wilkinson J.E., Park Y., Tuveson D.A, & Krainer A.R. (2023). Splicing Factor SRSF1 Promotes Pancreatitis and KRASG12D-Mediated Pancreatic Cancer. Cancer discovery, 13(7), 1678-1695.
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10

Western Blot Protein Analysis

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Cells were lysed in RIPA buffer containing protease inhibitors (cOmplete protease inhibitor cocktail, 11,836,170,001, Roche). Protein extracts were quantified using the Protein Assay Dye Reagent (5,000,006, BioRad) and 20 μg were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Cytiva Amersham). Membranes were blocked for 1 h in 5% milk in PBS-T (PBS with 0.1% Tween 20) and incubated overnight with the corresponding primary antibody in PBS-T 5% milk. For probing antibodies against TRP53 (NCL-L-p53-CM5p, Leica Biosystems), TP53 (sc-126, SCBT), p-γH2AX (9718 T, CST), βACTIN (sc-47778, SCBT), His-Tag (66,005–1-Ig, ThermoFisher), and secondary antibodies anti-rabbit (sc2357, SCBT), and anti-mouse (sc-516102, SCBT) were used. Membranes were developed using the ECL Prime system (RPN2232, Cytiva) and imaged using a ChemiDoc MP (BioRad).
Abad E., Sandoz J., Romero G., Zadra I., Urgel-Solas J., Borredat P., Kourtis S., Ortet L., Martínez C.M., Weghorn D., Sdelci S, & Janic A. (2024). The TP53-activated E3 ligase RNF144B is a tumour suppressor that prevents genomic instability. Journal of Experimental & Clinical Cancer Research : CR, 43, 127.
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