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47833
ATM Substrates Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit
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ATM Substrates Antibody Sampler Kit #47833

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Simple Western™ analysis of lysates (1 mg/mL) from COS-7 cells using p53 (7F5) Rabbit mAb #2527. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells treated with UV (100 J/m2 followed by a 3 hour recovery) and either Phospho-p53 (Ser15) (E9Y4U) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CDKN1A Promoter Primers #6449 and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Immunoprecipitation of Rad50 protein from PANC-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Rad50 (E3I8K) Rabbit mAb. Western blot analysis was performed using Rad50 (E3I8K) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Simple Western™ analysis of lysates (0.1 mg/mL) from 293T cells using Rad50 (E3I8K) Rabbit mAb #86225. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
Immunoprecipitation of phospho-Rad50 (Ser635) protein from 293 cell extracts. Cells were treated with UV (100 mJ, 4 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Rad50 (Ser635) (F5H8B) Rabbit mAb. Western blot analysis was performed using Phospho-Rad50 (Ser635) (F5H8B) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Flow cytometric analysis of untreated MCF7 cells (blue) and MCF7 cells treated with neocarzinostatin (0.5 µg/mL, 1 hr; green) using Phospho-Rad50 (Ser635) (F5H8B) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Western blot analysis of extracts from 293 and COS cells, using p53 (7F5) Rabbit mAb.
Western blot analysis of extracts from 293 cells, untreated or UV-treated (100 mJ/cm2, 1 hr), using Phospho-p95/NBS1 (Ser343) Antibody (upper) or p95/NBS1 (E8M3Q) XP® Rabbit mAb #81234 (lower).
Western blot analysis of extracts from control HeLa cells (lane 1) or HeLa cells with a targeted mutation in the gene encoding Chk2 (lane 2) using Chk2 (D9C6) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The change in Chk2 molecular weight in the mutated HeLa cells confirms the specificity of the antibody for Chk2.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Immunohistochemical analysis of paraffin-embedded human esophageal carcinoma using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Immunoprecipitation of p95/NBS1 protein from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is p95/NBS1 (E8M3Q) XP® Rabbit mAb. Western blot analysis was performed using p95/NBS1 (E8M3Q) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody. 
Western blot analysis of extracts from various human cell lines using p95/NBS1 (E8M3Q) XP® Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Negative expression of p95/NBS1 protein in GM07166 cells is consistent with the predicted expression pattern.
Western blot analysis of extracts from HCT 116 cells, untreated (-) or treated with neocarzinostatin (0.5 µg/mL, 1 hr; +), using Phospho-Chk2 (Thr68) (E8Q1A) Rabbit mAb (upper), Chk2 (D9C6) XP® Rabbit mAb #6334 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from A549 cells, mock treated (-) or treated with Doxorubicin #5927 (0.5 μM, 24 hr; +), using Phospho-p53 (Ser15) (E9Y4U) Rabbit mAb (upper), p53 (7F5) Rabbit mAb #2527 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Prior to blocking, membranes were either untreated (-) or treated with Calf Intestinal Phosphatase (CIP) (250 units/ml, 2 hr; +) at 37ºC.
Western blot analysis of extracts from various cell lines using Rad50 (E3I8K) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Low expression of Rad50 protein in LS 180 and SK-MEL-30 cells is consistent with the predicted expression pattern.
Western blot analysis of extracts from 293, ACHN, and C6 cells, untreated (-) or treated with UV (100 mJ, 4 hr, 4 hr, and 2 hr respectively; +), using Phospho-Rad50 (Ser635) (F5H8B) Rabbit mAb (upper), Rad50 Antibody #3427 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Phosphorylation of Rad50 protein at Ser635 is induced by UV treatment as expected.
Flow cytometric analysis of MCF7 cells treated with neocarzinostatin (0.5 µg/mL, 1 hr), and then treated post-fixation with λ phosphatase (blue) or untreated (green), using Phospho-Rad50 (Ser635) (F5H8B) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using p53 (7F5) Rabbit mAb.
Western blot analysis of extracts from various cell lines using Chk2 (D9C6) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human urothelial carcinoma using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or UV-treated (right), using Phospho-Chk2 (Thr68) (E8Q1A) Rabbit mAb.
Immunoprecipitation of phospho-p53 (Ser15) protein from A549 cell extracts treated with Doxorubicin #5927 (0.5 μM, 24 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-p53 (Ser15) (E9Y4U) Rabbit mAb. Western blot analysis was performed using Phospho-p53 (Ser15) (E9Y4U) Rabbit mAb. Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702 was used as a secondary antibody.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Rad50 (hRad50-Myc/DDK; +), using Rad50 (E3I8K) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western blot analysis of extracts from MCF7, 293T, and HCT 116 cells, untreated (-) or treated with Neocarzinostatin (0.5 μg/mL, 1 hr; +), using Phospho-Rad50 (Ser635) (F5H8B) Rabbit mAb (upper), Rad50 (E3I8K) Rabbit mAb #86225 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Phosphorylation of Rad50 protein at Ser635 is induced by neocarzinostatin treatment as expected.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using p53 (7F5) Rabbit mAb.
Immunoprecipitation of Chk2 from 293 cell extracts using Chk2 (D9C6) Rabbit mAb (lane 2). Western blot detection was performed using the same antibody. Lane 1 is 10% input.
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-Chk2 (Thr68) (E8Q1A) Rabbit mAb.
Confocal immunofluorescent analysis of A549 cells (p53-positive), untreated (first column), treated with Doxorubicin #5927 (0.5 µM, 24 hr; second column) to induce p53 phosphorylation, or treated with Doxorubicin and post-processed with λ-phosphatase (third column) to verify phospho-specificity. Saos-2 cells (p53-negative) were treated with Doxorubicin to verify specificity for p53 (fourth column). Cells were labeled with Phospho-p53 (Ser15) (E9Y4U) Rabbit mAb (green) and DyLight™ 554 Phalloidin #13054 (red) before mounting in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Immunohistochemical analysis of paraffin-embedded HT-29 (left) and SaOs-2 (right) cells, using p53 (7F5) Rabbit mAb. Note the lack of staining in p53-negative SaOs-2 cells.
Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Phospho-Chk2 (Thr68) (E8Q1A) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Confocal Immunofluorescent analysis of HT-29 cells using p53 (7F5) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
Immunohistochemical analysis of paraffin-embedded human B-cell non-Hodgkin lymphoma using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using Phospho-Chk2 (Thr68) (E8Q1A) Rabbit mAb.
Flow cytometric analysis of HT-29 cells using p53 (7F5) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human ovarian clear cell carcinoma, untreated (left) or lambda phosphatase-treated (right), using Phospho-Chk2 (Thr68) (E8Q1A) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human T-cell lymphoma using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Flow cytometric analysis of HeLa cells, untreated (blue) or treated with UV (100 mJ/cm2, 2 hr recovery; green), using Phospho-Chk2 (Thr68) (E8Q1A) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells treated with UV (100 J/m2 followed by a 3 hour recovery) and either p53 (7F5) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CDKN1A Promoter Primers #6449, human MDM2 intron 2 primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded normal human kidney using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded normal human thymus using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using p95/NBS1 (E8M3Q) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right). 
Immunohistochemical analysis of paraffin-embedded SK-MEL-28 cell pellet (left, positive) or GM07166 cell pellet (right, negative) using p95/NBS1 (E8M3Q) XP® Rabbit mAb.
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To Purchase # 47833
Cat. # Size Qty. Price
47833T		 1 Kit  (8 x 20 microliters)

Bulk & Custom Formulation

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Chk2 (Thr68) (E8Q1A) Rabbit mAb 82263 20 µl
  • WB
  • IHC
  • F
 H 62 Rabbit IgG
Chk2 (D9C6) Rabbit mAb 6334 20 µl
  • WB
  • IP
 H 62 Rabbit IgG
Phospho-p53 (Ser15) (E9Y4U) Rabbit mAb 82530 20 µl
  • WB
  • IP
  • IF
  • ChIP
 H 53 Rabbit IgG
p53 (7F5) Rabbit mAb 2527 20 µl
  • WB
  • IHC
  • IF
  • F
  • ChIP
 H Mk 53 Rabbit IgG
Rad50 (E3I8K) Rabbit mAb 86225 20 µl
  • WB
  • IP
 H 153 Rabbit IgG
Phospho-p95/NBS1 (Ser343) Antibody 3001 20 µl
  • WB
 H 95 Rabbit 
p95/NBS1 (E8M3Q) XP® Rabbit mAb 81234 20 µl
  • WB
  • IP
  • IHC
 H 95 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
 Rab Goat 
Phospho-Rad50 (Ser635) (F5H8B) Rabbit mAb 99215 20 µl
  • WB
  • IP
  • F
 H R 153 Rabbit IgG

Product Description

The ATM Substrates Antibody Sampler Kit provides an economical means to evaluate the signaling of ATM to downstream substrates. The kit contains enough primary and secondary antibodies to perform two Western blot experiments per primary antibody.

Background

ATM (ataxia telangiectasia mutated kinase) is a serine/threonine protein kinase best known for its role in DNA repair signaling in response to DNA double-strand breaks (DSBs). When DSBs occur, the MRE11:RAD50:NBS1 (MRN) sensor complex recruits ATM to sites of DNA damage. ATM then signals to numerous effector proteins, leading to cellular responses including regulation of DNA repair, cell cycle progression, apoptosis, senescence, gene transcription. Along with ATR, DNA-PKcs, SMG1 and mTOR, ATM is a member of the PI3K-like protein kinase (PIKK) family. PIKK family members typically function in response to various types of cellular stress. Substrates of ATM are numerous, and include CHK2, AKT, p53, BRCA1 and DNA-PK (reviewed in 1,3). Inactive ATM exists as a homodimer. In response to DSBs, ATM undergoes autophosphorylation in trans at Ser1981, which leads to dissociation of the complex to become an active monomer (2). Functional DNA repair pathways are important in cellular homeostasis, and defects in these pathways cause genomic instability, which can lead to tumorigenesis (3). Inactivation of ATM results in ataxia telangiectasia (AT), a neurodegenerative disease characterized by predisposition to cancer (4).

Pathways

Explore pathways related to this product.

Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
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U.S. Patent No. 5,675,063.
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