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Epigenetics and Nuclear Signaling DNA / RNA DNA Synthesis Other

Anti-MCM3抗体(ab4460)

  • Datasheet
  • SDS
Reviews (2)Q&A (2)References (23)

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Western blot - Anti-MCM3 antibody (ab4460)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCM3 antibody (ab4460)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCM3 antibody (ab4460)
  • Immunoprecipitation - Anti-MCM3 antibody (ab4460)
  • Western blot - Anti-MCM3 antibody (ab4460)
  • Western blot - Anti-MCM3 antibody (ab4460)
  • Immunocytochemistry/ Immunofluorescence - Anti-MCM3 antibody (ab4460)

Key features and details

  • Rabbit polyclonal to MCM3
  • Suitable for: ICC/IF, IP, WB, IHC-P
  • Reacts with: Mouse, Human
  • Isotype: IgG

选择批间可重复性更高的重组抗体

Product image
Anti-MCM3 antibody [EPR7080] (ab128923)
  • 研究可靠 —— 各批次间结果一致且可重复
  • 长期批量供应 —— 采用重组技术,可实现快速生产
  • 首次实验即可成功 —— 经过大量验证确认了特异性
  • 符合伦理标准 —— 产品不含动物成分

概述

  • 产品名称

    Anti-MCM3抗体
    参阅全部 MCM3 一抗
  • 描述

    兔多克隆抗体to MCM3
  • 宿主

    Rabbit
  • 经测试应用

    适用于: ICC/IF, IP, WB, IHC-Pmore details
  • 种属反应性

    与反应: Mouse, Human
    预测可用于: Orangutan
  • 免疫原

    Synthetic peptide within Human MCM3 aa 500-600. The exact immunogen sequence used to generate this antibody is proprietary information. If additional detail on the immunogen is needed to determine the suitability of the antibody for your needs, please contact our Scientific Support team to discuss your requirements. NP_002379.2 (GeneID 4172).
    Database link: P25205

  • 阳性对照

    • WB: HeLa cell RIPA extract. HeLa OHIO extract. IHC-P: Human colon carcinoma tissue. ICC/IF: HeLa cells.
  • 常规说明

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

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. Avoid freeze / thaw cycle.
  • 存储溶液

    pH: 7
    Preservative: 0.1% Sodium azide
    Constituents: 0.021% PBS, 1.764% Sodium citrate, 1.815% Tris
  • Concentration information loading...
  • 纯度

    Immunogen affinity purified
  • 克隆

    多克隆
  • 同种型

    IgG
  • 研究领域

    • Epigenetics and Nuclear Signaling
    • DNA / RNA
    • DNA Synthesis
    • Other

相关产品

  • ChIP Related Products

    • ChIP Kit (ab500)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab4460于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
ICC/IF
Use a concentration of 1 µg/ml.
IP
Use at 2-10 µg/mg of lysate.
WB (2)
1/2000 - 1/10000. Detects a band of approximately 105 kDa (predicted molecular weight: 90 kDa).
IHC-P
1/1000 - 1/5000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
说明
ICC/IF
Use a concentration of 1 µg/ml.
IP
Use at 2-10 µg/mg of lysate.
WB
1/2000 - 1/10000. Detects a band of approximately 105 kDa (predicted molecular weight: 90 kDa).
IHC-P
1/1000 - 1/5000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

靶标

  • 功能

    Acts as component of the MCM2-7 complex (MCM complex) which is the putative replicative helicase essential for 'once per cell cycle' DNA replication initiation and elongation in eukaryotic cells. The active ATPase sites in the MCM2-7 ring are formed through the interaction surfaces of two neighboring subunits such that a critical structure of a conserved arginine finger motif is provided in trans relative to the ATP-binding site of the Walker A box of the adjacent subunit. The six ATPase active sites, however, are likely to contribute differentially to the complex helicase activity. Required for DNA replication and cell proliferation.
  • 序列相似性

    Belongs to the MCM family.
    Contains 1 MCM domain.
  • 翻译后修饰

    Phosphorylated upon DNA damage, probably by ATM or ATR.
  • 细胞定位

    Nucleus.
  • Target information above from: UniProt accession P25205 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: 4172 Human
    • Entrez Gene: 17215 Mouse
    • Omim: 602693 Human
    • SwissProt: P25205 Human
    • SwissProt: P25206 Mouse
    • Unigene: 179565 Human
    • Unigene: 4502 Mouse
    • 别名

      • Cervical cancer proto oncogene 5 antibody
      • DNA polymerase alpha holoenzyme associated P1 antibody
      • DNA polymerase alpha holoenzyme associated protein P1 antibody
      • DNA polymerase alpha holoenzyme-associated protein P1 antibody
      • DNA replication factor MCM3 antibody
      • DNA replication licensing factor mcm3 antibody
      • HCC 5 antibody
      • HCC5 antibody
      • hRlf beta subunit antibody
      • Human cervical cancer proto oncogene 5 antibody
      • MCM 3 antibody
      • mcm3 antibody
      • MCM3 minichromosome maintenance deficient 3 antibody
      • MCM3_HUMAN antibody
      • MGC1157 antibody
      • Minichromosome maintenance complex component 3 antibody
      • Minichromosome maintenance deficient 3 antibody
      • Minichromosome maintenance protein 3 antibody
      • P1 h antibody
      • P1 MCM3 antibody
      • P1 Protein antibody
      • P1-MCM3 antibody
      • P1.h antibody
      • p102 antibody
      • P102 protein antibody
      • Replication licensing factor beta subunit antibody
      • RLF beta subunit antibody
      • RLF subunit beta antibody
      • RLFB antibody
      see all

    图片

    • Western blot - Anti-MCM3 antibody (ab4460)
      Western blot - Anti-MCM3 antibody (ab4460)

      Whole cell lysate (10 µg) from HeLa, Jurkat, HEK-293T, and TCMK-1 cells prepared using NETN lysis buffer.

      ab4460 used for WB at 0.04 µg/ml.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCM3 antibody (ab4460)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCM3 antibody (ab4460)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human small cell lung cancer tissue labelling MCM3 with ab4460 at 1/1000 (1µg/ml). Detection: DAB.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCM3 antibody (ab4460)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCM3 antibody (ab4460)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon carcinoma tissue labelling MCM3 with ab4460 at 1/1000 (1µg/ml). Detection: DAB.
    • Immunoprecipitation - Anti-MCM3 antibody (ab4460)
      Immunoprecipitation - Anti-MCM3 antibody (ab4460)

      Samples: Whole cell lysate (1.0 mg per IP reaction; 20% of IP loaded) from HeLa cells prepared using NETN lysis buffer.

      Lanes 1 and 2: ab4460 used for IP at 6 µg per reaction, two lots tested.

      Lane 3 Control IgG.

      For blotting immunoprecipitated MCM3, ab4460 was used at 0.04 µg/ml.

    • Western blot - Anti-MCM3 antibody (ab4460)
      Western blot - Anti-MCM3 antibody (ab4460)
      HeLa cell RIPA extract (50 µg) incubated with ab4460 used at 0.2 µg/ml for 1 hour. HeLa cell RIPA extract (50 µg) incubated with ab4460 used at 0.2 µg/ml for 1 hour.
    • Western blot - Anti-MCM3 antibody (ab4460)
      Western blot - Anti-MCM3 antibody (ab4460)
      WB using ab4460. Western blot analysis of ab4460 on HeLa OHIO extract.
    • Immunocytochemistry/ Immunofluorescence - Anti-MCM3 antibody (ab4460)
      Immunocytochemistry/ Immunofluorescence - Anti-MCM3 antibody (ab4460)
      ICC/IF image of ab4460 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab4460, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

    实验方案

    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    数据表及文件

    • SDS download

    • Datasheet download

      Download

    文献 (23)

    发表研究结果有使用 ab4460?请让我们知道,以便我们可以引用本数据表中的参考文章。

    ab4460 被引用在 23 文献中.

    • Drosopoulos WC  et al. TRF2 Mediates Replication Initiation within Human Telomeres to Prevent Telomere Dysfunction. Cell Rep 33:108379 (2020). PubMed: 33176153
    • Prikrylova T  et al. 5-hydroxymethylcytosine Marks Mammalian Origins Acting as a Barrier to Replication. Sci Rep 9:11065 (2019). PubMed: 31363131
    • Yang Q  et al. Minichromosome maintenance 3 promotes hepatocellular carcinoma radioresistance by activating the NF-?B pathway. J Exp Clin Cancer Res 38:263 (2019). PubMed: 31208444
    • Zhang Y  et al. Histone H3K27 methylation modulates the dynamics of FANCD2 on chromatin to facilitate NHEJ and genome stability. J Cell Sci 131:N/A (2018). PubMed: 29760279
    • Charrasse S  et al. Ensa controls S-phase length by modulating Treslin levels. Nat Commun 8:206 (2017). PubMed: 28785014
    View all Publications for this product

    客户评价及客户问答

    Show All 评价 Q&A
    提交评价 提交问题

    1-4 of 4 Abreviews or Q&A

    Western blot abreview for Anti-MCM3 antibody - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa)
    Loading amount
    50 µg
    Specification
    HeLa
    Gel Running Conditions
    Reduced Denaturing (12%)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    提交于 Nov 08 2011

    Western blot abreview for Anti-MCM3 antibody - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa)
    Loading amount
    25 µg
    Specification
    HeLa
    Gel Running Conditions
    Reduced Denaturing (12%)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Dr. Mirjam Eckert

    Verified customer

    提交于 Oct 30 2009

    Question

    a couple of month back I ordered one of your antibodies, anti-MCM3 antibody – ChIP Grade (ab4460). It was announced to be suitable for Western Blotting as well as immunoprecipitation. In Western Blotting it works fantastically, but for some reason I´ve got stuck with co-immunprecipitation. Could you please send me the protocol or some further information, how you tested the immunoprecipitation with this antibody?

    I´m trying to couple this antibody to beads and immunoprecipitate the MCM complex as well as putative interacting proteins. I have an MCM7-antibody as a positive control to check if I pulled down the whole complex.

    I´m using Dynabeads ® (invitrogen) cause when I used agarose beads MCM3 as well as MCM7 bound unspecifically to the beads. By Ponceau S staining I could see that the antibodies were coupled to the beads, as I could see the bands for the heavy and light chains of the antibodies, but I couldn´t detect the MCM7 in the pull-down of MCM3. So either my bait doesn´t bind to the antibody or the bait does bind and only the complex is not stable enough and MCM7 is not bound to MCM3 anymore. Could you maybe help with some recommendations or suggestions how to do the IP? Could it be that the antibody bind denatured protein but not the native one, as it works perfectly for Western Blotting?

    Read More

    Abcam community

    Verified customer

    Asked on Nov 01 2012

    Answer

    Thank you for contacting us.
    I'm sorry to hear that the antibody does not seem to work in IP.

    The protocol used to test the antibody in IP is as follows:

    Preparation of Cell Lysate used for Immunoprecipitation

    Reagents needed:

    PBS (Phosphate Buffered Saline, pH 7.2)


    NaCl 8.0 g
    KCl 0.2 g
    NaH2PO4 0.23 g
    Na2HPO4 0.12 g



    Adjust the volume to 1 liter with distilled water.

    Store at 4-25 C.

    IP Lysis Buffer


    NaCl 7.31 g
    1 M Tris, pH 8 25 ml
    0.5 M EDTA, pH 8 5 ml
    10% NP-40 25 ml
    Distilled Water 445 ml



    Store at 4 C.

    Protease Inhibitors
    Reconstitute with 1 ml of dH2O. Use 50 mcl per 5 ml of lysis buffer to harvest cells.

    Procedure:

    Allow cells to grow to approximately 80% confluence on 100mm polystyrene tissue culture plates. Approximately 8*10^6 cells (one plate at 80% confluence) are needed per IP reaction.
    Wash cells two times in cold PBS.
    Lyse cells to release soluble cellular proteins using 500 mcl of cold lysis buffer containing 1X protease inhibitors per 100 mm plate. Scrape cells from the plates, transfer to 1.5 ml micro-centrifuge tubes, and place on ice for 30 minutes to ensure efficient lysis.
    Centrifuge lysates at 10,000 x g; 5 minutes.
    Remove and pool the supernatant. Determine protein concentration.




    Immunoprecipitation Protocol

    Reagents Needed:

    Immunoprecipitation (IP) lysis buffer
    Protease Inhibitors
    Primary Antibodies
    Normal IgG, negative control
    Protein A Sepharose Beads
    Cell Lysate
    Sample Buffer

    IP lysis Buffer
    12.5 ml 1M NaCl (250mM)
    2.5 ml 1M Tris (50mM)
    500ul 0.5M EDTA (5mM)
    2.5ml 10% NP-40
    32 ml dH20

    Protein A Beads
    Resuspend 400 mg of Protein A beads in 10 ml of distilled H2O. Mix well to resuspend. Spin at 250 rpm for 5 minutes. Wash 3X in 10 ml IP Lysis buffer. Resuspend to 10 ml with IP lysis buffer for a 20% solution. Use 100 mcl per IP reaction.

    4X Sample Buffer
    Glycerol 4.0 g
    Tris Base 0.68 g
    Tris HCL 0.67 g
    LDS 0.80 g
    EDTA 6 mg
    Brilliant Blue G250 2.5 mg
    Phenol red 2.5 mg
    Adjust volume to 10 ml with ultra pure water.

    Store at 4 C.

    1X Sample Buffer
    4X sample buffer 150 mcl
    1M DTT 60 mcl
    Distilled water 390 mcl

    Make fresh for each use.

    Procedure:

    Prepare cell lysate according to protocol. Place 500 mcl of the prepared cell lysate (1-3 mg/ml) into a 1.5 ml micro-centrifuge tube.
    To this tube add 2 to 10 mcg of the primary antibody (If using neat sera or an IgG fraction such as Protein-A purified antibody, larger amounts are likely to be required. For best results, optimal amounts of antibody should be empirically defined.)
    To a negative control reaction, add an equivalent amount of normal rabbit IgG.
    Add 100 mcl of a 20% Protein A suspension to the mixture of antibody and cell lysate. Rotate the immunoprecipitation reactions (end-to-end) for 3 hours at room temperature or overnight at 4 C.
    Centrifuge (200 x g; 5 minutes) to pellet the complex.
    Remove the supernatant and add 500 mcl cold cell lysis buffer. Centrifuge (200 x g; 5 minutes).
    Repeat wash step 6 twice more. After each centrifugation remove as much of the supernatant as possible.
    After removing the supernatant from the third wash, add 40 mcl of freshly prepared 1X sample buffer to each tube and heat at 90 C for 5 minutes.
    Continue with electrophoresis and immunoblotting as described under western blotting protocol. Load 8 to 16 mcl (20 to 40% of the IP reaction) to a polyacrylamide gel.



    Note: For optimal results, complete reduction of the sample is required. We recommend the use of 0.1 M DTT in SDS-PAGE sample buffer and immediately heating samples, loading and running gels.

    Some additional advice and suggestions:

    The antibody has been tested without conjugation to beads, thus I would suggest trying it that way as well. If the conjugation does not work efficiently, then the IP will not work either.

    It is also important to include the isotype control (ab37415, https://www.abcam.com/Rabbit-IgG-ChIP-Grade-ab37415.html) as negative control as this would give you a better idea of the non-specific background.
    I'd suggest trying various antibody-to-lysate ratios (e.g. 1/100 to 1/1000) as the optimal ratio needs to be determined empirically and can vary from lysate type to lysate type and from antibody to antibody.
    Furthermore, in case that this might be a problem, for avoiding the heavy and light chain bands in the WB, I'd recommend using protein A/G-HRP (ab7456, ab7460) instead of the secondary antibody in the WB.

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    Read More

    Abcam Scientific Support

    回复于 Nov 01 2012

    Question

    I have a question about your MCM3 polyclonals - based on the possible sequence identity of the peptides that you use in your immunizations, do you expect any of your MCM3 antibodies to possibly recognize the Drosophila MCM3 protein? Thanking in advance,

    Read More

    Abcam community

    Verified customer

    Asked on Aug 08 2007

    Answer

    Thank you for your enquiry. There is a very low probability that ab3724 would react with Drosophila MCM3 and very low to zero probability that ab3725 or ab4460 would react too. I hope this information will be useful. Should you require any other assistance, please do not hesitate to contact me.

    Read More

    Abcam Scientific Support

    回复于 Aug 08 2007

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

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