概述

  • 产品名称
    Anti-ATM抗体[2C1 (1A1)]
    参阅全部 ATM 一抗
  • 描述
    小鼠单克隆抗体[2C1 (1A1)] to ATM
  • 宿主
    Mouse
  • 特异性
    The ATM antibody, clone 2C1, recognizes full-length ATM.
  • 经测试应用
    适用于: Flow Cyt, ICC/IF, IHC-P, WB, IPmore details
  • 种属反应性
    与反应: Mouse, Rat, Human, Monkey
  • 免疫原

    Fusion protein expressed in E. coli corresponding to amino acids 2577-3056.

  • 阳性对照
    • lymphoblastoid nuclear lysate, human Raji, U87-MG , SK-N-SH (human neuroblastoma), IMR32 , SK-N-AS.
  • 常规说明

    This product was changed from ascites to tissue culture supernatant on 23rd October 2018. Please note that the dilutions may need to be adjusted accordingly. If you have any questions, please do not hesitate to contact our scientific support team

性能

应用

Our Abpromise guarantee covers the use of ab78 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
Flow Cyt Use 1-2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF Use at an assay dependent concentration.
IHC-P Use a concentration of 1 µg/ml.
WB 1/2000. Detects a band of approximately 350 kDa (predicted molecular weight: 350 kDa).
IP Use a concentration of 1 - 10 µg/ml.

靶标

  • 功能
    Serine/threonine protein kinase which activates checkpoint signaling upon double strand breaks (DSBs), apoptosis and genotoxic stresses such as ionizing ultraviolet A light (UVA), thereby acting as a DNA damage sensor. Recognizes the substrate consensus sequence [ST]-Q. Phosphorylates 'Ser-139' of histone variant H2AX/H2AFX at double strand breaks (DSBs), thereby regulating DNA damage response mechanism. Also plays a role in pre-B cell allelic exclusion, a process leading to expression of a single immunoglobulin heavy chain allele to enforce clonality and monospecific recognition by the B-cell antigen receptor (BCR) expressed on individual B lymphocytes. After the introduction of DNA breaks by the RAG complex on one immunoglobulin allele, acts by mediating a repositioning of the second allele to pericentromeric heterochromatin, preventing accessibility to the RAG complex and recombination of the second allele. Also involved in signal transduction and cell cycle control. May function as a tumor suppressor. Necessary for activation of ABL1 and SAPK. Phosphorylates p53/TP53, FANCD2, NFKBIA, BRCA1, CTIP, nibrin (NBN), TERF1, RAD9 and DCLRE1C. May play a role in vesicle and/or protein transport. Could play a role in T-cell development, gonad and neurological function. Plays a role in replication-dependent histone mRNA degradation. Binds DNA ends.
  • 组织特异性
    Found in pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, heart, spleen, thymus, testis, ovary, small intestine, colon and leukocytes.
  • 疾病相关
    Defects in ATM are the cause of ataxia telangiectasia (AT) [MIM:208900]; also known as Louis-Bar syndrome, which includes four complementation groups: A, C, D and E. This rare recessive disorder is characterized by progressive cerebellar ataxia, dilation of the blood vessels in the conjunctiva and eyeballs, immunodeficiency, growth retardation and sexual immaturity. AT patients have a strong predisposition to cancer; about 30% of patients develop tumors, particularly lymphomas and leukemias. Cells from affected individuals are highly sensitive to damage by ionizing radiation and resistant to inhibition of DNA synthesis following irradiation.
    Note=Defects in ATM contribute to T-cell acute lymphoblastic leukemia (TALL) and T-prolymphocytic leukemia (TPLL). TPLL is characterized by a high white blood cell count, with a predominance of prolymphocytes, marked splenomegaly, lymphadenopathy, skin lesions and serous effusion. The clinical course is highly aggressive, with poor response to chemotherapy and short survival time. TPLL occurs both in adults as a sporadic disease and in younger AT patients.
    Note=Defects in ATM contribute to B-cell non-Hodgkin lymphomas (BNHL), including mantle cell lymphoma (MCL).
    Note=Defects in ATM contribute to B-cell chronic lymphocytic leukemia (BCLL). BCLL is the commonest form of leukemia in the elderly. It is characterized by the accumulation of mature CD5+ B lymphocytes, lymphadenopathy, immunodeficiency and bone marrow failure.
  • 序列相似性
    Belongs to the PI3/PI4-kinase family. ATM subfamily.
    Contains 1 FAT domain.
    Contains 1 FATC domain.
    Contains 1 PI3K/PI4K domain.
  • 结构域
    The FATC domain is required for interaction with KAT5.
  • 翻译后修饰
    Phosphorylated by NUAK1/ARK5. Autophosphorylation on Ser-367, Ser-1893, Ser-1981 correlates with DNA damage-mediated activation of the kinase.
    Acetylation, on DNA damage, is required for activation of the kinase activity, dimer-monomer transition, and subsequent autophosphorylation on Ser-1981. Acetylated in vitro by KAT5/TIP60.
  • 细胞定位
    Nucleus. Cytoplasmic vesicle. Primarily nuclear. Found also in endocytic vesicles in association with beta-adaptin.
  • Information by UniProt
  • 数据库链接
  • 别名
    • A-T mutated antibody
    • A-T mutated homolog antibody
    • AT mutated antibody
    • AT1 antibody
    • ATA antibody
    • Ataxia telangiectasia mutated antibody
    • Ataxia telangiectasia mutated gene antibody
    • Ataxia telangiectasia mutated homolog (human) antibody
    • Ataxia telangiectasia mutated homolog antibody
    • ATC antibody
    • ATD antibody
    • ATDC antibody
    • ATE antibody
    • ATM antibody
    • ATM serine/threonine kinase antibody
    • ATM_HUMAN antibody
    • DKFZp781A0353 antibody
    • MGC74674 antibody
    • OTTHUMP00000232981 antibody
    • Serine protein kinase ATM antibody
    • Serine-protein kinase ATM antibody
    • Serine/threonine-protein kinase ATM antibody
    • Tefu antibody
    • TEL1 antibody
    • TEL1, telomere maintenance 1, homolog antibody
    • TELO1 antibody
    • Telomere fusion protein antibody
    see all

图片

  • All lanes : Anti-ATM antibody [2C1 (1A1)] (ab78) at 1/500 dilution

    Lane 1 : 30ug HeLa
    Lane 2 : 30ug HeLa nuclear extract
    Lane 3 : 30ug SK-N-SH

    Predicted band size: 350 kDa

  • ab78 staining ATM in Human Testis sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Samples were incubated with primary antibody (5ug/ml) and a Biotin-conjugated rabbit anti-mouse IgG was used as the secondary antibody.

  • ab78 staining ATM in Human U2OS cells by Immunocytochemistry/ Immunofluorescence.

    Cells were fixed with paraformaldehyde, permeabilized with 0.5% NP40/PBS and blocked with 3% BSA for 1 hour at 25°C. Samples were incubated with primary antibody (1/100 in 3% BSA/PBS) for 12 hours at 4°C. An AlexaFluor®488-conjugated goat anti-rabbit polyclonal IgG (1/200) (ab150077) was used as the secondary antibody.

  • Overlay histogram showing HeLa cells stained with ab78 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab78, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • All lanes : Anti-ATM antibody [2C1 (1A1)] (ab78) at 1/2000 dilution

    Lane 1 : 50ug lymphoblastoid nuclear lysate, anti-beta actin antibody.
    Lane 2 : 25ug lymphoblastoid nuclear lysate, anti-beta actin antibody.
    Lane 3 : No ATM negative control, anti-beta actin antibody.

    Secondary
    All lanes : Anti-mouse secondary antibody at 1/2000 dilution

    Performed under reducing conditions.

    Predicted band size: 350 kDa



    Arrows denote the location of the 45kDa beta actin protein, and the 350kDa ATM protein.
  • ab78 (2µg/ml) staining ATM in human colonic mucosa, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining of mucosal epithelium and lymphocytes.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

  • Detection of human ATM protein using anti-ATM 2C1 monoclonal antibody (ab78) by western blot.

  • ab78 staining ATM in Human Kidney sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Samples were incubated with primary antibody (5ug/ml) and a Biotin-conjugated rabbit anti-mouse IgG was used as the secondary antibody.

  • Detection of human ATM protein using anti-ATM 2C1 monoclonal antibody (ab78) by immunoprecipitation.

文献

This product has been referenced in:
  • Collin G  et al. Transcriptional repression of DNA repair genes is a hallmark and a cause of cellular senescence. Cell Death Dis 9:259 (2018). Read more (PubMed: 29449545) »
  • Ling X  et al. TERT regulates telomere-related senescence and apoptosis through DNA damage response in male germ cells exposed to BPDE in vitro and to B[a]P in vivo. Environ Pollut 235:836-849 (2018). Mouse . Read more (PubMed: 29353801) »
See all 54 Publications for this product

客户评价及客户问答

1-10 of 21 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Cow Tissue sections (Ovarian cortical strips)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Natrium citrate
Permeabilization
No
Specification
Ovarian cortical strips
Blocking step
(agent) for 1 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 24°C
Fixative
Formaldehyde

Mrs. Mila Maidarti

Verified customer

提交于 Apr 06 2017

Application
Western blot
Loading amount
10 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (MCF7)
Specification
MCF7
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

提交于 Feb 12 2015

Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris)
Sample
Human Cell lysate - nuclear (HeLa nuclear cell lysate)
Specification
HeLa nuclear cell lysate
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

提交于 Nov 20 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (HEK293)
Specification
HEK293
Treatment
Doxorubicin for 3 hours
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Dr. Andrew Cobb

Verified customer

提交于 Jul 11 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (U2OS)
Specification
U2OS
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% NP40/PBS
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

提交于 Aug 27 2012

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued an alternativefree of charge replacement of ab2618:

Order number: 1042580
1 X FOC vial ab2618

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Question

Hi Kate,

I have attached the completed questionnaire form. Thank you for your help.

Kind regards,

Order Details

Antibody code:
ab78 -Mouse monoclonal [2C1 (1A1)] to ATM

Lot number GR64682-2

General Information
Antibody storage conditions (temperature/reconstitution etc)
Antibody was aliquoted and stored at -20oC as described in the technical data sheet.

Description of the problem (high background, wrong band size, more bands, no band etc.)
High background, signal appears as a smear rather than clear distinct bands.


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Whole cell extracts of various mouse cell lines


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)

Cell pellets re-suspended in SDS loading buffer, heated at 95oC, sonicated and heated again.

Amount of protein loaded
Whole cell extract used (represents amount of protein in 200,000 cells)


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
6% SDS-PAGE gel


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Wet transfer at 4°C for 1.5 hours. Membranes blocked in 5% milk at RT for 1 hour.

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
ab78 -Mouse monoclonal [2C1 (1A1)] to ATM
1:1,000 dilution used
Incubated at 4oC overnight in 5% milk
Washed in PBST 3 X 10 mins

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

ImmunoPure® HRP conjugated goat anti-rabbit IgG antibody (Thermo Scientific)
1:1,000
Incubated at RT for 1 hour
Washed in PBST 3 x 10 mins

Detection method (ECL, ECLPlus etc.)
SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific)

Positive and negative controls used (please specify)

I wanted to detect the basal expression levels of ATM in my cell lines – no positive/negative control required

Optimization attempts (problem solving)
How many times have you tried the Western?

Three times

Have you run a "No Primary" control?
Yes

Do you obtain the same results every time?
Yes
e.g. are the background bands always in the same place?


What steps have you altered?

Running samples on 6% SDS PAGE gel for longer period of time
Running samples on 4% SDS PAGE gels

Additional Notes:

Read More
Answer

Thank you for taking the time to complete our questionnaire.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimize the results from ab78. I would also appreciate if you can confirm some further details:

1. I would appreciate if you are able to provide an image, including molecular weight markers, which would help me to assess the results.

2. Please confirm which mouse cell lines have been tested?

3. I can suggest the cells need to be fully lysed in lysis buffer such as RIPA. SDS loading buffer alone will not necessarily provide good lysis and protein preparation.

4. I can also suggest toinclude protease inhibitors in the lysis buffer as smearing down the lane on the blot indicates the proteins in the sample may have degraded. For the same reason, I can suggest to try fresh samples and reagents if possible.

5. I recommend to check the amount of protein being loaded, to ensure the gel is not overloaded. We recommend to do a protein assay on each sample and to load 20 - 30 ug of total protein per lane of the gel.

6. Smearing may also indicate the gel has been run too quickly, particularly with a larger protein. Try reducing the voltage and increasing the gel running time which should help provide a better resolution.

7. Reducing the concentration of antibody will help to reduce any background. Try 1:2000 as recommended on the datasheet.

8. I can recommend these further tips for transfer of larger proteins, from our protocols pages:

Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation.
Lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily.
Methanol is only necessary if using nitrocellulose. If using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich.

9. Could you confirm ifthe secondary isworking well with other primary antibodies? What are the results of the no primary control?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested information and I will be pleased to provide the alternative free of charge replacment

Read More

Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for western blotting, and for Mouse, Rat, Human and Monkey samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund. Alteratively, as requested we can arrange a free of charge replacment of an alternative antibody.

Although the other antibodies have worked well using this procedure, individual antibodies will often require optimization. Before deciding how to proceed in this case, I would like to investigate this further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.



Order Details
Antibody code:
ab78 -Mouse monoclonal [2C1 (1A1)] to ATM


Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:

Read More

Answer

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab78with the order number 1009642. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

Read More

Question
Answer

Thank you for your enquiry. The Abcam lab runs Invitrogen pre-cast SDS-PAGE gels at 200 volts for 35mins, and runs the transfer for 60 mins at 30 volts. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Read More

1-10 of 21 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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