Whole-cell protein lysates of treated cells were extracted and fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose as previously described 14 (link). Protein expression was determined using the Western Lightning Plus-ECL detection system (NEL104001EA, PerkinElmer, MA, USA). Primary antibodies used: γ-H2AX (ab2893, abcam), activated caspase3 (ab32042, abcam), CHK2 pThr 68 (2661, Cell Signaling), CHK2 (3440, Cell Signaling), ATM pSer 1981 (5883, Cell Signaling) and GAPDH (MAB374, Millipore). Secondary antibodies used: anti-rabbit IgG, HRP-linked antibody (7074, Cell Signaling) and anti-mouse IgG, HRP-linked antibody (7076, Cell Signaling).
Atm pser 1981
Manufactured by Cell Signaling Technology
Sourced in United States
The ATM pSer 1981 is a laboratory product that detects phosphorylation of the ATM protein at serine residue 1981. It is used as a tool for studying the activation of the ATM protein, which is a key regulator of the cellular response to DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
Chk2 pthr 68, by Cell Signaling Technology (3 mentions)
Ab2893, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Mab374, by Merck Group (1 mentions)
Ecl substrate, by Merck Group (1 mentions)
Ne per extraction reagents kit, by Thermo Fisher Scientific (1 mentions)
P53 pser15, by Cell Signaling Technology (1 mentions)
Tris hcl gel, by Bio-Rad (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
X ray film, by Fujifilm (1 mentions)
β actin a2066, by Merck Group (1 mentions)
Ab5821, by Abcam (1 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (1 mentions)
Img 124a, by Novus Biologicals (1 mentions)
H 300, by Merck Group (1 mentions)
Bu 33, by Merck Group (1 mentions)
5 protocols using atm pser 1981
1
Western Blot Analysis of DNA Damage Response
2
Western Blot Analysis of DNA Damage Response
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Protein content was measured with BCA assay (Pierce), 5–30 µg was subjected to SDS/PAGE on 4–20% acrylamide gel (Thermo Scientific) followed by transfer to nitrocellulose membrane. When detecting ATM, 7.5% Tris-HCl gel (Bio-Rad) was used, and protein was transferred to PVDF membrane (Millipore) overnight at 30 V. Membranes were blocked with 5% milk in TBS-T (Tween 0.1% wt/vol), and probed with the following antibodies: p53 pSer15 (1∶1000, Cell Signaling), p53 (1∶1000, Cell Signaling), ATM pSer1981 (1∶1000, Cell Signaling), α-tubulin (1∶2000, Santa Cruz), ERK2 (1∶2000, Santa Cruz), dCK (Clone 9D4, 1∶1000, Millipore). Polyclonal rabbit antibody against dCK pSer74 was a gift from Dr. Francoise Bontemps (Universite Catholique de Louvain, Brussels, Belgium). ECL substrate (Millipore) was used for detection and development on GE/Amersham film. For separating nuclear and cytoplasmic lysates, NE-PER Extraction Reagents kit was used (Thermo Scientific).
3
Immunoblot Analysis of Signaling Pathways
4
Protein Profiling by Western Blot
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Cell lysates, SDS-PAGE and Western blot were performed as described in [5 (link)]. Antibodies used were: ATM p-Ser1981 #13050, Chk2 p-Thr68 #2661, Chk2 #2662, Chk1 p-Ser345 #2341, Chk1 #2360, KAP1 #5868, cleaved caspase 3 (Asp175) #9661 (Cell Signaling Technology, Danvers, MA, USA), KAP1 p-Ser473 #644602 (Biolegend, San Diego, CA, USA), ATM #AF1655 (R&D Systems Bio-Techne, Minneapolis, MN, USA), vinculin #V9131, β-actin #A2066 (Millipore Sigma, Burlington, MA, USA). Detection was performed by exposure to standard X-ray films or by a CCD camera system (UVITEC, Cambridge, UK).
5
Immunofluorescence Analysis of Cellular Markers
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Antibodies included fibrillarin 1∶1000 (Abcam ab18380 or ab5821), CENP-A 1∶500 (Abcam 13939 or Upstate 07-574), TRF2 1∶200 (Imgenex IMG-124A or Novus NB110-57130), Ki67 antigen 1∶500 (Novocastra Laboratories Ltd.), H2AX-p 1∶300 (Millipore 05-636 or Abcam ab2893), ATM-p Ser1981 1∶250 (Cell Signaling 5883), Chk2-p Thr68 1∶250 (Cell Signaling 2661), SMC2 1∶300 (Cell Signaling 5394), SMC4 1∶300 (Cell Signaling 5547), goat polyclonal to DDDDK (FLAG) tag 1∶500 (Abcam ab1257), UBF 1∶150 (Santa Cruz H-300), and anti-BrdU 1∶400 (Sigma clone BU-33). Primary antibodies were detected using anti-mouse, anti-rabbit, or anti-goat secondary antibodies conjugated to Alexa Fluor 488, 594, 647 (Molecular Probes), FITC, Cy3, or Cy5 (Jackson Immunoresearch, Inc.).
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