Overview

  • Product name
  • Description
    Rabbit polyclonal to Survivin
  • Host species
    Rabbit
  • Specificity
    Specific for survivin.
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-P, ELISA, Flow Cyt, IP, RIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Cat, Dog, Human
  • Immunogen

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

  • Positive control
    • WB: Jurkat whole cell lysate (ab7899), HeLa whole cell lysate (ab150035). ICC: HeLa cells. IHC-P: Human ovarian carcinoma, rectal cancer and colon carcinoma tissues.

Properties

Applications

Our Abpromise guarantee covers the use of ab469 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
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

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

  • 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.

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

  • 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).

  • ab469 staining Survivin from 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

  • 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. 

  • 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

  • ab469 immunoprecipitating Survivin from HeLa cell lysate. HeLa cells were lysed after colcemid block (ON) and immunoprecipitated with ab469 at a 1/1000 dilution. HeLa cells clear lysate (Lane 1) as well as the bound material (Lane 2) were loaded on a 15 % acrylamide gel. An HRP conjugated Donkey Anti-rabbit IgG was used as the secondary antibody.

    This image is courtesy of an Abreview by Sandrine Ruchaud submitted on 12 April 2006.

    See Abreview

  • 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
  • Paraformaldehyde-fixed, paraffin-embedded human colon carcinoma tissue stained for Survivin using ab469 at 1/500 dilution in immunohistochemical analysis.

    See Abreview

References

This product has been referenced in:
  • Yan H  et al. NR4A1-induced increase in the sensitivity of a human gastric cancer line to TNFa-mediated apoptosis is associated with the inhibition of JNK/Parkin-dependent mitophagy. Int J Oncol 52:367-378 (2018). Read more (PubMed: 29207128) »
  • Lu C  et al. Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR-424-3p and targeting PTEN/PI3K/Akt pathway. J Cell Mol Med N/A:N/A (2018). Read more (PubMed: 29392841) »
See all 75 Publications for this product

Customer reviews and Q&As

1-10 of 31 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Jul 04 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Dr. Eleni Petsalaki

Verified customer

Submitted Oct 08 2012

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,

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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

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

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

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/index.html?datasheet=24479).  

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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.

Read More

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 guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Dec 17 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Mar 26 2009

1-10 of 31 Abreviews or Q&A

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