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Cell Biology Apoptosis Intracellular Survivin / IAPs

Anti-Survivin抗体(ab469)

  • Datasheet
Reviews (10)Q&A (21)References (119)

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Western blot - Anti-Survivin antibody (ab469)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Survivin antibody (ab469)
  • Immunocytochemistry/ Immunofluorescence - Anti-Survivin antibody (ab469)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Survivin antibody (ab469)
  • Immunocytochemistry/ Immunofluorescence - Anti-Survivin antibody (ab469)
  • Immunocytochemistry/ Immunofluorescence - Anti-Survivin antibody (ab469)
  • Western blot - Anti-Survivin antibody (ab469)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Survivin antibody (ab469)

Key features and details

  • Rabbit polyclonal to Survivin
  • Suitable for: ICC/IF, WB, IHC-P, ELISA, Flow Cyt, IP, RIP
  • Reacts with: Mouse, Rat, Cow, Cat, Dog, Human
  • Isotype: IgG

选择批间可重复性更高的重组抗体

Product image
Anti-Survivin antibody [EP2880Y] (ab76424)
  • 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-Survivin抗体
    参阅全部 Survivin 一抗
  • 描述

    兔多克隆抗体to Survivin
  • 宿主

    Rabbit
  • 经测试应用

    适用于: ICC/IF, WB, IHC-P, ELISA, Flow Cyt, IP, RIPmore details
  • 种属反应性

    与反应: Mouse, Rat, Cow, Cat, Dog, Human
  • 免疫原

    Recombinant full length protein corresponding to Human Survivin .
    Database link: O15392

  • 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 long term. Avoid freeze / thaw cycle.
  • 存储溶液

    pH: 7.40
    Preservative: 0.05% Sodium azide
    Constituents: 0.876% Sodium chloride, 99% Tris glycine
  • Concentration information loading...
  • 纯度

    Immunogen affinity purified
  • 克隆

    多克隆
  • 同种型

    IgG
  • 研究领域

    • Cell Biology
    • Apoptosis
    • Intracellular
    • Survivin / IAPs
    • Neuroscience
    • Cell Type Marker
    • Glia marker
    • Astrocyte marker
    • Neuroscience
    • Neurology process
    • Neurodegenerative disease
    • Other
    • Neuroscience
    • Neurology process
    • Neurogenesis
    • Cancer
    • Invasion/microenvironment
    • Apoptosis
    • Death receptors & ligands
    • IAPs
    • Cancer
    • Cell Death
    • Apoptosis
    • Receptors
    • Death receptors & ligands
    • IAPs

相关产品

  • 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)
  • Positive Controls

    • Jurkat whole cell lysate (ab7899)
    • A-431 whole cell lysate (ab7909)
  • Recombinant Protein

    • Recombinant Human Survivin protein (ab87202)

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab469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 (2)
1/250. See Abreview by William Moore; fix with formaldehyde.
WB (4)
Use a concentration of 1 µg/ml. Predicted molecular weight: 16 kDa. Found to work at 1/5000 dilution.
IHC-P (3)
Use a concentration of 0.5 µg/ml. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
ELISA
Use at an assay dependent concentration.
Flow Cyt
Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

IP (1)
Use at an assay dependent concentration.

Recommended to use at 5-7µg/ml.

RIP
Use at an assay dependent concentration. PubMed: 19542185
说明
ICC/IF
1/250. See Abreview by William Moore; fix with formaldehyde.
WB
Use a concentration of 1 µg/ml. Predicted molecular weight: 16 kDa. Found to work at 1/5000 dilution.
IHC-P
Use a concentration of 0.5 µg/ml. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
ELISA
Use at an assay dependent concentration.
Flow Cyt
Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

IP
Use at an assay dependent concentration.

Recommended to use at 5-7µg/ml.

RIP
Use at an assay dependent concentration. PubMed: 19542185

靶标

  • 功能

    Component of the chromosomal passenger complex (CPC), a complex that acts as a key regulator of mitosis. The CPC complex has essential functions at the centromere in ensuring correct chromosome alignment and segregation and is required for chromatin-induced microtubule stabilization and spindle assembly. The complex with RAN plays a role in mitotic spindle formation by serving as a physical scaffold to help deliver the RAN effector molecule TPX2 to microtubules. May play a role in neoplasia. May counteract a default induction of apoptosis in G2/M phase. Inhibitor of caspase-3 and caspase-7. Isoform 2 and isoform 3 do not appear to play vital roles in mitosis. Isoform 3 shows a marked reduction in its anti-apoptotic effects when compared with the displayed wild-type isoform.
  • 组织特异性

    Expressed only in fetal kidney and liver, and to lesser extent, lung and brain. Abundantly expressed in adenocarcinoma (lung, pancreas, colon, breast, and prostate) and in high-grade lymphomas. Also expressed in various renal cell carcinoma cell lines.
  • 序列相似性

    Belongs to the IAP family.
    Contains 1 BIR repeat.
  • 发展阶段

    Expression is cell cycle-dependent and peaks at mitosis.
  • 结构域

    The BIR repeat is necessary and sufficient for HBXIP binding.
  • 翻译后修饰

    Ubiquitination is required for centrosomal targeting.
    In vitro phosphorylation at Thr-117 by AURKB/STK12 prevents interaction with INCENP and localization to mitotic chromosomes.
  • 细胞定位

    Cytoplasm. Nucleus. Chromosome. Chromosome > centromere. Cytoplasm > cytoskeleton > spindle. Localizes on chromosome arms and inner centromeres from prophase through metaphase and then transferring to the spindle midzone and midbody from anaphase through cytokinesis. Colocalizes with AURKB at mitotic chromosomes.
  • Target information above from: UniProt accession O15392 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: 493835 Cat
    • Entrez Gene: 414925 Cow
    • Entrez Gene: 442936 Dog
    • Entrez Gene: 332 Human
    • Entrez Gene: 11799 Mouse
    • Entrez Gene: 64041 Rat
    • Omim: 603352 Human
    • SwissProt: Q6I6F4 Cat
    • SwissProt: Q6J1J1 Cow
    • SwissProt: Q8I009 Dog
    • SwissProt: O15392 Human
    • SwissProt: O70201 Mouse
    • SwissProt: Q9JHY7 Rat
    • Unigene: 514527 Human
    • Unigene: 8552 Mouse
    • Unigene: 54471 Rat
    see all
  • 别名

    • API4 antibody
    • Apoptosis inhibitor 4 antibody
    • Apoptosis inhibitor survivin antibody
    • Apoptosis inhibitor4 antibody
    • Baculoviral IAP repeat containing 5 antibody
    • Baculoviral IAP repeat containing protein 5 antibody
    • Baculoviral IAP repeat-containing protein 5 antibody
    • BIRC 5 antibody
    • BIRC5 antibody
    • BIRC5_HUMAN antibody
    • EPR 1 antibody
    • IAP4 antibody
    • Survivin variant 3 alpha antibody
    • SVV antibody
    • TIAP antibody
    see all

图片

  • Western blot - Anti-Survivin antibody (ab469)
    Western blot - Anti-Survivin antibody (ab469)
    All lanes : Anti-Survivin antibody (ab469) at 1 µg/ml

    Lane 1 : HeLa Nuclear
    Lane 2 : HeLa whole cell lysate
    Lane 3 : A431 cell lysate
    Lane 4 : Jurkat cell lysate
    Lane 5 : HEK293 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Alexa Fluor anti-rabbit at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 16 kDa
    Observed band size: 18 kDa why is the actual band size different from the predicted?
    Additional bands at: 37 kDa, 50 kDa. We are unsure as to the identity of these extra bands.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Survivin antibody (ab469)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Survivin antibody (ab469)

    Paraffin-embedded human rectal cancer tissue stained for Survivin using ab469 at 0.5 µg/ml in immunohistochemical analysis, using DAB with hematoxylin counterstain.

  • Immunocytochemistry/ Immunofluorescence - Anti-Survivin antibody (ab469)
    Immunocytochemistry/ Immunofluorescence - Anti-Survivin antibody (ab469)

    HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained for Survivin (green) using ab469 at 1/10 dilution in ICC/IF. An Alexa Fluor 488-conjugated Goat to rabbit IgG was used as secondary antibody (green). Actin filaments were labeled with Alexa Fluor 568 phalloidin (red). DAPI was used to stain the cell nuclei (blue).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Survivin antibody (ab469)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Survivin antibody (ab469)This image is courtesy of an Abreview submitted by Dr Ben Davidson

    ab469 staining Survivin from Human Ovarian carcionoma tumour tissue sections by Immunohistochemistry (Formalin-fixed paraffin-embedded sections). Heat mediated antigen retrieval was performed (Citrate buffer pH=6, microwave oven) and the tissue was then formaldehyde fixed and blocked (Hydrogen peroxide 0.03%). An HRP conjugated goat anti-rabbit was used as the secondary antibody.

    See Abreview

  • Immunocytochemistry/ Immunofluorescence - Anti-Survivin antibody (ab469)
    Immunocytochemistry/ Immunofluorescence - Anti-Survivin antibody (ab469)This image is courtesy of William Moore

    HeLa cells (ab150035) in prometaphase, metaphase and anaphase stained with anti-Survivin (green), anti-tubulin (red) and DAPI (blue). These images were kindly supplied as part of the review submitted by William Moore, University of Dundee, UK. 

    See Abreview

  • Immunocytochemistry/ Immunofluorescence - Anti-Survivin antibody (ab469)
    Immunocytochemistry/ Immunofluorescence - Anti-Survivin antibody (ab469)

    ab469 at a 1/400 dilution staining HeLa cells by Immunocytochemistry. The antibody was incubated with the cells for 1 hour and then was detected using a Texas Red conjugated Goat anti-rabbit antibody.

    This image is courtesy of an Abreview by Sandrine Ruchaud submitted on 30 March 2006.

    See Abreview

  • Western blot - Anti-Survivin antibody (ab469)
    Western blot - Anti-Survivin antibody (ab469)
    Anti-Survivin antibody (ab469) at 1 µg/ml + HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 30 µg

    Developed using the ECL technique.

    Predicted band size: 16 kDa


    Exposure time: 1 minute
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Survivin antibody (ab469)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Survivin antibody (ab469)This image is courtesy of an Abreview submitted by Mr. Rudolf Jung.
    Paraformaldehyde-fixed, paraffin-embedded human colon carcinoma tissue stained for Survivin using ab469 at 1/500 dilution in immunohistochemical analysis.

    See Abreview

实验方案

  • Recommended protocols with ab469

Click here to view the general protocols

数据表及文件

  • Datasheet download

    Download

文献 (119)

发表研究结果有使用 ab469?请让我们知道,以便我们可以引用本数据表中的参考文章。

ab469 被引用在 119 文献中.

  • Mejia J  et al. A replicating stem-like cell that contributes to bone morphogenetic protein 2-induced heterotopic bone formation. Stem Cells Transl Med 10:623-635 (2021). PubMed: 33245845
  • Wang ZG  et al. Endogenous conversion of n-6 to n-3 polyunsaturated fatty acids facilitates the repair of cardiotoxin-induced skeletal muscle injury in fat-1 mice. Aging (Albany NY) 13:8454-8466 (2021). PubMed: 33714197
  • Zuco V  et al. Selinexor versus doxorubicin in dedifferentiated liposarcoma PDXs: evidence of greater activity and apoptotic response dependent on p53 nuclear accumulation and survivin down-regulation. J Exp Clin Cancer Res 40:83 (2021). PubMed: 33648535
  • Meng X  et al. miR-101-3p sensitizes lung adenocarcinoma cells to irradiation via targeting BIRC5. Oncol Lett 21:282 (2021). PubMed: 33732358
  • Martini S  et al. miR-34a-Mediated Survivin Inhibition Improves the Antitumor Activity of Selinexor in Triple-Negative Breast Cancer. Pharmaceuticals (Basel) 14:N/A (2021). PubMed: 34072442
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

1-10 of 31 Abreviews or Q&A

Western blot abreview for Anti-Survivin antibody

Excellent
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Mouse Cell lysate - whole cell (endothelial)
Gel Running Conditions
Reduced Denaturing (14)
Loading amount
10 µg
Specification
endothelial
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°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 Jul 04 2017

Western blot abreview for Anti-Survivin antibody

Good
Abreviews
Abreviews
Application
Western blot
Sample
Human Cell lysate - whole cell (Human colon carcinoma cells)
Loading amount
75 µg
Specification
Human colon carcinoma cells
Gel Running Conditions
Non-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.

DR. Eleni Petsalaki

Verified customer

提交于 Oct 08 2012

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Survivin antibody

Excellent
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (ovarian cancer)
Specification
ovarian cancer
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate, pH 6.0, 100C, 20 min
Permeabilization
No
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 Dec 17 2010

Western blot abreview for Anti-Survivin antibody

Excellent
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa cells)
Loading amount
50 µg
Specification
HeLa cells
Gel Running Conditions
Reduced Denaturing (12)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°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 Mar 26 2009

Question

Good afternoon, Thank you for your fast response to my e-mail. First aswering to the last two questions: I've been using Streptavidin protein (HRP) (ab7403) as for thedilution of the secondary antibody is 1/250 Nowwith regard to the form sent: 1) Abcam product code ab469 3) Description of the problem: Need more intense staining 4) Sample preparation: Species: Rat Type of sample:PFA/formalin fixed paraffin embedded sections Negative control: Removal of primary antibody 5) Fixation step Yes If yes: Fixative agent and concentration:paraformaldehyde 4% Fixation time: at least 48 hours Fixation temperature 4ºC 6) Antigen retrieval method: Heat-induced with 10mM Sodium Citrate Buffer 7) Permeabilization method: Not done 8) Blocking agent (eg BSA, serum…):Normal Goat serum Concentration 5% in TBST Blocking time: 1h Blocking temperature: room temperature 9) Endogenous peroxidases blocked? Yes (3% Hydrogen Peroxide) Endogenous biotins blocked? Yes (Endogenous avidin + biotin blocking system ab3387) 10) Primary antibody (If more than one was used, describe in “additional notes”) : Anti-Survivin (ab469) Concentration or dilution 1/500 Diluent buffer1xTBST Incubation time Overnight 11) Secondary antibody: Goat Polyclonal Secondary antibody to Rabbit IgG - H&L (Biotin) (ab6720) Concentration or dilution 1/250 Diluent buffer1xTBST Incubation time 2h 12) Washing after primary and secondary antibodies: Yes Buffer: 1x TBST Number of washes at least 3 times 13) Detection method: Streptavidin Protein (HRP) (ab7403) DAB substrate kit (ab94665) 14) How many times have you run this staining? Several Times Do you obtain the same results every time?yes almost every time What steps have you altered to try and optimize the use of this antibody? Altering the dilution of primary antibody, altering the time of incubation of the primary antibody, altering the time of incubation of the cromogen. Best regards,

Read More

Abcam community

Verified customer

Asked on Jan 04 2012

Answer

Thank you for providing that extra information. It has helped greatly to understand what you have been doing and what may help to improve the signal of the staining. There are a few areas that I would suggest would be worth optimising (if you have not already done so) which may lead to an improved signal. 1. Fixation You have stated that you are fixing the tissue for a minimum of 48 hours. The ideal fixation time will depend on the size of the tissue block and type of tissue but usually 18-24 hours seems ideal for most applications. Over-fixation can lead to the epitope being masked. 2. Antigen retrieval Antigen retrieval can be used to overcome the masking mentioned for the fixation. Depending on the level of masking, different conditions of antigen retrieval may be required. For example, if using a microwave we would usually suggest trying retrieval for 5, 10, 15 and 20 minutes to see which produces the optimal results. 3. Hydrogen peroxide blocking I would suggest performing the hydrogen peroxide blocking following the incubation of the primary antibody if you have not already tried this. The hydrogen peroxide can sometimes affect sensitive epitopes and by performing the step following the incubation with the primary antibody this will not affect the staining. 4. Primary antibody incubation You mention that you have optimised this step by changing the dilution and incubation time. If you have not already done so I would suggest attempting the incubation for 1-1.5 hours at room temperature with agitation. 5. Detection method Different techniques can be used to directly amplify the signal. Currently you are using streptavidin-HRP conjugate which is labelled in a 1:1 ratio. It is possible to employ avidin which has been labelled in a 3:1 ratio with HRP using kits such as Piercenet product 32020. Although avidin can cause greater background compared to using streptavidin. Alternatively HRP polymer can be employed. This consists of using a secondary antibody which is attached to a polymer-HRP complex. Abcam has the following product to offer, ab2891, however this is a little pricey. I would suggest trying to optimise the steps 1-4 and if sufficient progress is not made contemplate employing one of signal amplification methods mentioned. We have quite a detailed guide to IHC which may be of help to you. This outlines many of the different experimental parameters which need to be considered when performing IHC and suggestions to improve experiments: https://www.abcam.com/ps/pdf/protocols/ihc_p.pdf I hope this information has been of help and your staining improves. If you want any further information or help please do not hesitate to contact us again.

Read More

Abcam Scientific Support

回复于 Jan 04 2012

Question

Good morning, I'm currently using Abcam products to perform imunohistochemistry on paraffinon my research protocols and I have some doubts that I would likeAbcam tohelp me. I worked with Anti-Survivin antibody (ab469) as a primary antibody in a 1/500 dilution. I useda goat polyclonal secondary antibody (ab6720) as well as endogenous Avidin + Biotin Blocking System (ab3387) and DAB Substrate (ab94665)kit as a cromogen. With this experience Iwas able to get favorable results, however I would like to know how to get a more intense staining?I tried to optimize the dilution of primary antibody as well as the exposure time of DAB butcould not get significant changes. Could you give mean advise on how to get some more intense staining? Another question about the primary antibody Anti-Smac / Diablo (ab8114) is what dilution do you recomend using in IHC-P protocols? I read the datasheet where you say to use a concentration of 5ug/ml but I can't understand how to perform this concentration/ diluition. Best regards,

Read More

Abcam community

Verified customer

Asked on Jan 03 2012

Answer

Thank you for contacting us. I'm glad to hear you have been getting good staining with ab469. In order to help you obtain more intense staining would you mind filling in the form which I have attached to this email. It will allow me to more fully understand the protocol which you have been performing and any areas in which it may be worthwhile to optimise. Could you also answer the following questions: 1. which HRP conjugate have you been using (streptavidin-HRP? could you tell me the catalogue no. etc) 2. in what dilution have you been using the secondary antibody? ab8114 is supplied at a concentration of 1 mg/mL. The recommended working concentration of this antibody when using it for immunohistochemistry with paraffin embedded sections is 5 µg/ml. This equates to a dilution of 1/200. This is only a guideline and may need to be optimised for the staining which you are performing. I hope this information has been of help and look forward to your reply.

Read More

Abcam Scientific Support

回复于 Jan 03 2012

Question

We are using your rabbit polyclonal anti-survivin antibody (ab469).  According to the data sheet, the immunogen is "recombinant full length protein."  Do you know whether this anitbody is, therefore, specific only for the Wild Type, or does it also recognize other splice variants?  

Read More

Abcam community

Verified customer

Asked on Dec 08 2011

Answer

Thank you for contacting us. A similar question was asked by a customer in May of 2008. The question, which contains some references, and our reply, can be found under the Scientific Support tab of the online datasheet. Without testing reactivity with the splice variants, we cannot be sure which the antibody will detect. However, the splice variants listed in the UniProt database entry for the human survivin, accession O15392, (the URL is http://www.uniprot.org/uniprot/O15392) all share the same 73 N-terminal amino acids. Given that this antibody is raised against the full-length isoform 1, also know as alpha isoform, which inludes the 73-amino acid range, and assuming that the antibody recognizes at least one epitope in that range, the antibody should be capapble of recognizing all splice forms. However, depending on how the protein folds, epitopes in the N-terminal range may be inaccesible to the antibody. This should be considered a possibilty if you are trying to detect survivin in its native conformation, for instance by IHC or ELISA. The ability to make a consistent prediction of reactivity with variants is also complicated by the nature of polyclonal antibodies, insofar as the epitopes they recogize will vary with each immunization and the resulting sera. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Abcam Scientific Support

回复于 Dec 08 2011

Question

 Thanks for your help. The membranes were blotted with the GFP Ab and then stripped and re-probed with Ab-survivin. The gel on the right is in the absence of FBS (fetal bovine serum). I didn’t try a different GFP Ab. The fact that the anti-GFP pic a 17bp band in the cells overexpressing GFP only makes me think that band is not specific. Do you recommend a better surviving Ab?    

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Abcam community

Verified customer

Asked on Oct 17 2011

Answer

Thank you the clarification. The 17 kDa band is strange, insofar as it appears in the GFP-stained blot, looks very similar to the `17 kDa band in the survivn-stained blot, and appears only in the lanes containing transfected cell lysates, which argues against it being endogenous survivin. I think you may be getting cleavage of the survivin from the GFP. That would explain the 17 kDa band in the lanes of transfected samples in the survivin blot. I am still having trouble understanding the differences among your samples. In particular, can you tell me how YFP-CON differs from YFP-SURV? I am assuming that YFP-CON is just a transfection with YFP, based on the GFP signal in those lanes. But then there is no explanation for the 17 kDa survivin signal in those lanes, in the survivin blot. For an alternative survivin antibody, I suggest ab24479, which gives clean blots on a variety of samples. Click here (or use the following: https://www.abcam.com/Survivin-antibody-ab24479.html).  

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Abcam Scientific Support

回复于 Oct 17 2011

Question

 The Ab Lot number is ****. We use 5%milk to block the membrane and I tried boiled and not boiled samples. Please see attached ppt for the westerns done with different Abs. NT stands for not transfected. What loading buffer do you advise to enrich the monomer and get rid of the dimer.    

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Abcam community

Verified customer

Asked on Oct 17 2011

Answer

Thank you for sending the images. The blots stained with ab469 have an isolated band at 17 kDa which I assume is endogenous survivin, but that same band appears in the GFP-stained blots. Were the anti-GFP blots also stained with ab469? Also, what is the difference between the blots on the left from the ones on the right, in the ppt file you sent? What is the sample on the far right in the left-side blots? The images you send do make the problem more clear, though, than the earlier TIFF file. I do not think multimers are the problem, assuming you are adding a reducing agent in your loading buffer before boiling the sampes. What is interesteing is that the GFP antibody (if it is the only one being used to stain the upper blots) is picking up the same multiple bands as the survivin antibody, suggesting that your cells may be producing several truncated transcripts of different sizes. I cannot tell if the bands are the same size though, since the markers in the survivin blots are not labeled. Have you tried any other GFP antibodies? If you see the same pattern, then you may need to consider that possibility of multiple transcripts. I look forward to your reply, and to resolving this issue. It may require another antibody.

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Abcam Scientific Support

回复于 Oct 17 2011

Question

We ordered this antibody from abcam (ab469) a while ago but I doubt it is picking a specific band. Can you please help us with a feedback from tech support about this. I tried this Ab to detect over expressed GFP-survivin and to detect endogenous survivin. I always get two bands near the right size but several other higher bands. Attached please find an image of my last blot to detect surviving in a purified cellular compartment.

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Abcam community

Verified customer

Asked on Oct 13 2011

Answer

Thank you for contacting us about the western blot issue you are having with this antibody. We typically ask for a few details of the protocol to see if there are modifications we can suggest, but I expect you have a standard protocol that works well with other antibodies. We would also like to have the lot number of the antibody. It will be on the tube. If you do not have that, can you tell me the PO number or approximate date of the order? Some of the bands in the blot image appear to be multimers of the 16 kDa survivin monomer, along with other bands that may represent non-survivin proteins that the antibody cross-reacts with. We have not received similar reports. Can you please tell me what the samples are and how they are prepared for loading into the gel? What do you use for blocking the membrane, and is the secondary effective with other rabbit antibodies, with these samples? It is unlikely that the secondary is at fault but we ask, just in case it is a new antibody in the lab. Finally, which lanes contain the GFP-survivin, and which contain the un-transfected cell lysates? I look forward to your reply. If I cannot offer a suggestion, we will replace the antibody with a different lot or a different antibody altogether. If you prefer, we will also consider a credit or refund. I look forward to your reply.

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Abcam Scientific Support

回复于 Oct 13 2011

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