Recombinant
RabMAb

Recombinant Anti-Survivin antibody [EP2880Y] (ab76424)

Overview

  • Product name

    Anti-Survivin antibody [EP2880Y]
    See all Survivin primary antibodies
  • Description

    Rabbit monoclonal [EP2880Y] to Survivin
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, WB, IP, Flow Cyt, ICC/IF, Sandwich ELISAmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human Survivin aa 1-100 (N terminal). The exact sequence is proprietary.
    Database link: O15392

  • Positive control

    • HeLa and Jurkat lysates; human urinary bladder carcinoma and human colonic adenocarcinoma tissues; A549; Ramos; Human bladder carcinoma
  • General notes

    A trial size is available to purchase for this antibody.

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab76424 in the following tested applications.

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

Application Abreviews Notes
IHC-P 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Signal amplification system might be necessary or the use of polymerized HRP secondary antibodies.

For unpurified use at 1/250 - 1/500. 

WB 1/5000 - 1/20000. Detects a band of approximately 16 kDa (predicted molecular weight: 16 kDa).
IP 1/150.

For unpurified use at 1:10. 

Flow Cyt 1/500.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

For unpurified use at 1/50. 

ICC/IF 1/100 - 1/250.
Sandwich ELISA Use a concentration of 0.5 µg/ml. Can be paired for Sandwich ELISA with Goat polyclonal to Survivin (ab27468).

For sandwich ELISA, use this antibody as detection at 0.5µg/ml with ab27468 as capture.

Target

  • Function

    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.
  • Tissue specificity

    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.
  • Sequence similarities

    Belongs to the IAP family.
    Contains 1 BIR repeat.
  • Developmental stage

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

    The BIR repeat is necessary and sufficient for HBXIP binding.
  • Post-translational
    modifications

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

    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.
  • Information by UniProt
  • Database links

  • Alternative names

    • 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

Images

  • Anti-Survivin antibody [EP2880Y] (ab76424) at 1/5000 dilution (purified) + A549 (Human lung carcinoma epithelial cell) whole cell lysates at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 16 kDa



    Blocking and diluting buffer : 5% NFDM/TBST
  • ab76424 (purified) at 1:150 dilution (5µg) immunoprecipitating Survivin in Ramos whole cell lysate.
    Lane 1 (input): Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10µg
    Lane 2 (+): ab76424 & Ramos whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76424 in Ramos whole cell lysate
    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

  • Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Survivin with purified ab76424 at 1:500 dilution (5 µg/ml) (red). Cells were fixed with 80% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder carcinoma tissue sections labeling Survivin with Purified ab76424 at 1:1000 dilution (2.52 µg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using Tris/EDTA Buffer,PH9. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
    secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.
  • Immunofluorescent staining of MCF-7 cells labelling Bcl-2 with purified ab76424 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue). 

    Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

  • All lanes : Anti-Survivin antibody [EP2880Y] (ab76424) at 1/5000 dilution (purified)

    Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
    Lane 2 : Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysates

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 16 kDa



    Blocking and diluting buffer : 5% NFDM/TBST
  • All lanes : Anti-Survivin antibody [EP2880Y] (ab76424) at 1/10000 dilution (unpurified)

    Lane 1 : HeLa lysates
    Lane 2 : Jurkat lysates

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : goat anti-rabbit HRP at 1/1000 dilution

    Predicted band size: 16 kDa
    Observed band size: 16 kDa

  • Unpurified ab76424 showing positive staining in Fetal kidney tissue.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Overlay histogram showing HeLa cells stained with unpurified ab76424 (red line). The cells were fixed with 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 (ab76424, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized with 0.1% PBS-Tween 20 used under the same conditions.

  • Unpurified ab76424 at 1/250 dilution staining Survivin in human colonic adenocarcinoma by Immunohistochemistry, Paraffin-embedded tissue.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Unpurified ab76424 at 1/250 dilution staining Survivin in human urinary bladder carcinoma by Immunohistochemistry, Paraffin-embedded tissue.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Unpurified ab76424 showing positive staining in breast carcinoma tissue.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Unpurified ab76424 showing positive staining in cervical carcinoma tissue.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Unpurified ab76424 showing positive staining in fetal liver tissue.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Unpurified ab76424 showing positive staining in normal tonsil tissue.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling Survivin with unpurified ab76424 at 1/100. Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cells were incubated with the primary antibody overnight. An Alexa Fluor® 555-conjugated anti-rabbit IgG was used as the secondary antibody. Left - Survivin, Right - DAPI.

  • Standard curve for Survivin (Analyte: ab87202); dilution range 1pg/ml to 1µg/ml using Capture Antibody ab27468 at 1µg/ml and Detector Antibody abRabbit monoclonal [EP2880Y] to Survivin (ab76424) at 0.5µg/ml. Concentration of Rabbit monoclonal [EP2880Y] to Survivin (ab76424) may vary from lot to lot; please use this curve as guideline.
  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD
  • ab76424 staining Survivin in the Hela cell line from Human cervix by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. Ab150081 (1/200) was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:

  • Hou J  et al. TROP2 promotes the proliferation and metastasis of glioblastoma cells by activating the JAK2/STAT3 signaling pathway. Oncol Rep 41:753-764 (2019). Read more (PubMed: 30431125) »
  • Gao LM  et al. Tumor-suppressive effects of microRNA-181d-5p on non-small-cell lung cancer through the CDKN3-mediated Akt signaling pathway in vivo and in vitro. Am J Physiol Lung Cell Mol Physiol N/A:N/A (2019). Read more (PubMed: 30628487) »
See all 56 Publications for this product

Customer reviews and Q&As

1-6 of 6 Q&A

Question
Answer

Thank you very much for your call earlier this week, and I apologize for the delay in getting this information for you.

The antibody ab76424 was used at a 1:2000 dilution in sandwich ELISA.

I hope that this will be useful, but please let me know if you have any further questions and I'll be happy to help. Have a nice day!

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Answer

Thank you very much for your email.

xxx is currently not in the office but I will pass the message on. I'm sure she will be very pleased.

If we can be of any further assistance, please do not hesitate to let me know.

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Question

Product code: 76424
Lot number: GR81000-1
Inquiry:
1)Antibody storage conditions (temperature/reconstitution etc): the antibody was stored at -20oC
2)Description of the problem (high background, wrong band size, more bands, no band etc.): No band detection
3)Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.): whole cell lysate ZL55 e ZL34 cell lines
4)Sample preparation (Buffer/Protease inhibitors/Heating sample etc.): RIPA buffer plus Protease and Phosphatase inhibitors
5)Amount of protein loaded: 25 and 50 ug
6)Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.): Denaturing SDS PAGE (12%)
7)Transfer and blocking conditions (Buffer/time period, Blocking agent etc.): Semi-dry transfer Blocking: 5% milk prepared in TBS+Tween (0.01%)
8)Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):
Primary antibody: anti-survivin primary antibody (Abcam cat n° ab76424). diluition 1/2000 and 1/1000 in blocking buffer Incubation time: over night at 4°C Wash step: TBS+Tween for 10 minutes (three times)
9)Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): Secondary antibody: anti rabbit pierce diluition 1/2000 in blocking buffer Incubation time: 1 hour at room temperature Wash step: TBS+Tween for 10 minutes (three times)
10)Detection method (ECL, ECLPlus etc.): Supersignal West femto chemiluminescent substrate (Thermo Scientific).
11)Positive and negative controls used (please specify): whole lysate from HeLa cells

12)How many times have you tried the Western?: three times

13)Have you run a No Primary control?: no

The customer has no images because there was no signal at all.

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear the customer has had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will provide us with vital information for our monitoring of product quality. I would like to reassure you that this antibody is tested and covered by our guarantee for WB and human samples.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results. I can suggest you may have regrettably received a bad vial.

I can recommend you may like to consider trying the following options:

1. Could you confirm if the transfer to the membrane and quality of the sample has been assessed using a loading control?

2. Is the current vial of secondary antibody working well with other rabbit antibodies?

3. I can suggest to try using BSA rather than milk to block. Changing blocking agents can sometimes help to improve the results.

Alternatively, or if these tips do not work, I am pleased to offer you a free of charge replacement or credit note in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Question
Answer

Thank you for contacting Abcam regarding ab76424.


This antibody is predicted to cross-react with all 7 isoforms of survivin, based on 100% sequence homology.


I hope this information is helpful. Please let me know if you have any additional questions.

Read More

Answer

Thank you for your inquiry.

I can confirm that the immunogen maps to the first 25 amino acids of human Survivin, on the N-terminus (UniProt: O15392).

Therefore the antibody should recognise all isoforms since they do not differ in the first 25 amino acids according to the Swiss Prot website:

http://www.uniprot.org/uniprot/O15392

I hope this information is helpful for your customer. Please feel free to contact me again with any further questions.

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Answer

Thank you for contacting Abcam regarding ab76424 and ab3731. I am sorry that you have been experiencing difficulties getting these antibodies to work in WB.  I am following up with the lab regarding reactivity of ab76424 with Delta Ex3.  Regarding ab3731, one point to note is that this antibody only works well in Western blotting on cell lysates overexpressing DEx3(see PMID 16847456), and it first requires IP with another antibody, before blotting with this antibody. It has been previously used with an IP performed from 5 million cells overexpressing DEx3.  If you have not performed an IP, the antibody will not likely detect the protein in WB. I hope this information is helpful.  Please do not hesitate to contact us if you have any additional questions.

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