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Epigenetics and Nuclear Signaling DNA / RNA DNA Damage & Repair DNA Damage Response ATM / ATR

Anti-ATM抗体[2C1 (1A1)] - BSA and Azide free (ab78)

  • Datasheet
  • SDS
Reviews (8)Q&A (13)References (81)

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Western blot - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
  • Flow Cytometry - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
  • Western blot - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
  • Immunoprecipitation - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)

Key features and details

  • Mouse monoclonal [2C1 (1A1)] to ATM - BSA and Azide free
  • Suitable for: Flow Cyt, IHC-P, WB, IP
  • Reacts with: Human
  • Isotype: IgG1

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概述

  • 产品名称

    Anti-ATM抗体[2C1 (1A1)] - BSA and Azide free
    参阅全部 ATM 一抗
  • 描述

    小鼠单克隆抗体[2C1 (1A1)] to ATM - BSA and Azide free
  • 宿主

    Mouse
  • 特异性

    The ATM antibody, clone 2C1, recognizes full-length ATM.
  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    IHC-P
    Human
    IP
    Human
    WB
    Human
    See all applications and species data
  • 免疫原

    Fusion protein corresponding to ATM aa 2577-3056.
    Database link: Q13315

  • 阳性对照

    • WB: HeLa nuclear extract, lymphoblastoid nuclear lysate. IHC-P: Human Testis sections, human colonic mucosa, human kidney sections. ICC/IF: Human U2OS cells Flow: HeLa cells
  • 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

  • 形式

    Liquid
  • 存放说明

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • 存储溶液

    pH: 7.40
    Constituent: 100% PBS
  • 无载体

    是
  • Concentration information loading...
  • 纯化说明

    Purified from TCS by Protein G chromatography to at least 95% homogeneity as determined by SDS-PAGE.
  • 克隆

    单克隆
  • 克隆编号

    2C1 (1A1)
  • 骨髓瘤

    NS1
  • 同种型

    IgG1
  • 轻链类型

    kappa
  • 研究领域

    • Epigenetics and Nuclear Signaling
    • DNA / RNA
    • DNA Damage & Repair
    • DNA Damage Response
    • ATM / ATR
    • Cancer
    • Oncoproteins/suppressors
    • Tumor suppressors
    • Other

相关产品

  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
    • Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879)
  • Isotype control

    • Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control (ab170190)
    • Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control (ab91353)
  • Recombinant Protein

    • Recombinant Human ATM protein (ab131739)

应用

The Abpromise guarantee

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.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

应用 Species
Flow Cyt
Human
IHC-P
Human
IP
Human
WB
Human
All applications
Mouse
Rat
Monkey
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.

IHC-P (1)
Use a concentration of 5 µg/ml.
WB (5)
1/500 - 1/3000. Detects a band of approximately 350 kDa (predicted molecular weight: 350 kDa).
IP
Use a concentration of 1 - 10 µg/ml.
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.

IHC-P
Use a concentration of 5 µg/ml.
WB
1/500 - 1/3000. 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.
  • Target information above from: UniProt accession Q13315 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • 数据库链接

    • Entrez Gene: 472 Human
    • Entrez Gene: 11920 Mouse
    • Entrez Gene: 300711 Rat
    • Omim: 607585 Human
    • SwissProt: Q13315 Human
    • SwissProt: Q62388 Mouse
    • Unigene: 367437 Human
    • Unigene: 5088 Mouse
    • 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

    • Western blot - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
      Western blot - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
      All lanes : Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78) at 1/500 dilution

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

      Secondary
      All lanes : Mouse IgG antibody (HRP) at 1/5000 dilution

      Predicted band size: 350 kDa



      5% SDS-PAGE.

      Running conditions: 80V, 15min; 140V, 40 minutes.

      Transfer condition: Semi-dry, 18 V, 60 min (NC membrane).

      Blocking condition: 5% non-fat milk in TBST, RT, 60 minutes.

      Primary antibody incubation: 4°C, overnight.

      Washing condition: 5 ml TBST, 4 x 5 minutes. 

      Exposure: enhanced ECL

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)

      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.

    • Flow Cytometry - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
      Flow Cytometry - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)

      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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)

      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.

    • Western blot - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
      Western blot - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)

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

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)

      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.

    • Immunoprecipitation - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)
      Immunoprecipitation - Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)

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

    实验方案

    • Flow cytometry protocols
    • Immunoprecipitation protocols
    • Immunohistochemistry protocols
    • Western blot protocols

    Click here to view the general protocols

    数据表及文件

    • SDS download

    • Datasheet download

      Download

    文献 (81)

    发表研究结果有使用 ab78?请让我们知道,以便我们可以引用本数据表中的参考文章。

    ab78 被引用在 81 文献中.

    • Deng L  et al. p53-mediated control of aspartate-asparagine homeostasis dictates LKB1 activity and modulates cell survival. Nat Commun 11:1755 (2020). PubMed: 32273511
    • Wang J  et al. Inhibition of PLK4 might enhance the anti-tumour effect of bortezomib on glioblastoma via PTEN/PI3K/AKT/mTOR signalling pathway. J Cell Mol Med 24:3931-3947 (2020). PubMed: 32126150
    • Nam AR  et al. Therapeutic Targeting of the DNA Damage Response Using an ATR Inhibitor in Biliary Tract Cancer. Cancer Res Treat 51:1167-1179 (2019). PubMed: 30514066
    • Pang J  et al. Vorapaxar stabilizes permeability of the endothelial barrier under cholesterol stimulation via the AKT/JNK and NF-?B signaling pathways. Mol Med Rep 19:5291-5300 (2019). PubMed: 31059055
    • Jin MH  et al. Therapeutic Co-targeting of WEE1 and ATM Downregulates PD-L1 Expression in Pancreatic Cancer. Cancer Res Treat N/A:N/A (2019). PubMed: 31291716
    View all Publications for this product

    客户评价及客户问答

    Show All 评价 Q&A
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    1-10 of 21 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-ATM antibody [2C1 (1A1)]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Bos taurus Cell (Ovarian follicle, granulosa cells)
    Permeabilization
    Yes - 0.1% PBST
    Specification
    Ovarian follicle, granulosa cells
    Blocking step
    (agent) for 1 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 23°C
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    提交于 Feb 17 2020

    Western blot abreview for Anti-ATM antibody [2C1 (1A1)]

    Poor
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa and Lymphoblastoid)
    Gel Running Conditions
    Reduced Denaturing (3-8% Tris-Acetate)
    Loading amount
    10 µg
    Specification
    HeLa and Lymphoblastoid
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Read More

    Ellie Crompton

    Verified customer

    提交于 Aug 12 2019

    Western blot abreview for Anti-ATM antibody [2C1 (1A1)]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (PNT2 prostate tissue)
    Gel Running Conditions
    Reduced Denaturing (6)
    Loading amount
    30 µg
    Specification
    PNT2 prostate tissue
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More

    Abcam user community

    Verified customer

    提交于 Jan 08 2019

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-ATM antibody [2C1 (1A1)]

    Good
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Mrs. Mila Maidarti

    Verified customer

    提交于 Apr 06 2017

    Western blot abreview for Anti-ATM antibody [2C1 (1A1)]

    Excellent
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    提交于 Feb 12 2015

    Western blot abreview for Anti-ATM antibody [2C1 (1A1)]

    Excellent
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    提交于 Nov 20 2013

    Western blot abreview for Anti-ATM antibody [2C1 (1A1)]

    Good
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Dr. Andrew Cobb

    Verified customer

    提交于 Jul 11 2013

    Immunocytochemistry/ Immunofluorescence abreview for Anti-ATM antibody [2C1 (1A1)]

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    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    提交于 Aug 27 2012

    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

    Abcam community

    Verified customer

    Asked on Feb 27 2012

    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

    Abcam Scientific Support

    回复于 Feb 27 2012

    Question

    Dear Sir/Madam,

    I recently purchased the following Abcamantibody:

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

    I have tried this antibody several times on various samples for Western Blotting but the results have been very unsatisfactory as I am unable to detect a clear signal for ATM. The same membranes were re-probed with antibodies against ATR and Beta-Tubulin and these proteins were detected without any problems (please see attached powerpoint slide).

    I would like to enquire whether I canorder another anti-ATM antibody free of charge as a replacement:

    ab2618: Anti-ATM antibody [5C2]

    If this is not possible, I would like to request a refund for the ab78 anti-ATM antibody that was previously purchased.

    Kind regards,

    Read More

    Abcam community

    Verified customer

    Asked on Feb 24 2012

    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

    Abcam Scientific Support

    回复于 Feb 24 2012

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