Anti-Hsp90 antibody [AC88] (ab13492)

Mouse monoclonal Hsp90 antibody [AC88]. Validated in WB, IP, IHC, ICC, Flow Cyt, ICC/IF and tested in Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Hamster, Cow, Dog, Human, Pig and more.

Overview

  • Product name
    Anti-Hsp90 antibody [AC88]
    See all Hsp90 primary antibodies
  • Description
    Mouse monoclonal [AC88] to Hsp90
  • Host species
    Mouse
  • Tested applications
    Suitable for: IHC-Fr, IHC-P, ICC/IF, WB, IP, Flow Cyt, ICCmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Hamster, Cow, Dog, Human, Pig, Caenorhabditis elegans, Carp, Fish, Monkey, African green monkey, Rainbow trout
  • Immunogen

    Full length native protein (purified) corresponding to Hsp90. from Achlya ambisexualis (water mold).

  • Epitope
    The epitope of this antibody has been mapped to amino acid residues 604-697 of the human Hsp90 sequence.
  • Positive control
    • ICC/IF: Panc-1 and HepG2 cells. WB: HeLa Cell Lysate (Heat Shocked), Hsp90 Protein; HepG2 cell lysate; PC12 cell lysate; NIH/3T3 cell lysate; Rat2 cell lysate. IHC-P: Human spleen and testis tissue. IHC-fr: Human stomach tissue.
  • General notes

    This product was changed from ascites to tissue culture supernatant on 22nd May 2019. 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.

Properties

  • Form
    Liquid
  • Storage instructions
    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.
  • Storage buffer
    pH: 7.20
    Preservative: 0.09% Sodium azide
    Constituents: 49% PBS, 50% Glycerol
  • Concentration information loading...
  • Purity
    Protein G purified
  • Clonality
    Monoclonal
  • Clone number
    AC88
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab13492 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-Fr Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 90 kDa.

84.7 (alpha) , 83.2  (beta)

IP Use at an assay dependent concentration.

When used in immunoprecipitation, this antibody does not appear to react well with Hsp90 that is bound to steroid receptors or to pp60v-src of Rouse sarcoma virus.

Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC Use at an assay dependent concentration.

Target

Images

  • ab13492 staining Hsp90 (red) and another antibody to C2GnT-M (Golgi enzyme, greeen) in Panc-1 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with PFA and blocked with 1% serum for 1 hour at 22°C. Samples were incubated with primary antibody (1/50 1% Donkey serum in PBST) for 1 hour at 22°C. An undiluted DyLight® 594-conjugated Donkey anti-mouse IgG polyclonal was used as the secondary antibody.

    This image was generated using the ascites version of the product. 

    See Abreview

  • All lanes : Anti-Hsp90 antibody [AC88] (ab13492) at 1/1000 dilution

    Lane 2 : HSP90 cell lysate
    Lane 3 : HSP90ß cell lysate
    Lane 4 : HSP90a cell lysate
    Lane 5 : HeLa (heat shocked) cell lysate
    Lane 6 : NIH/3T3 (Heat shocked) cell lysate
    Lanes 7-8 : PC-12 (Heat shocked) cell lysate


    This image was generated using the ascites version of the product.

  • Anti-Hsp90 antibody [AC88] (ab13492) at 1/1000 dilution + Human fibroblast whole cell lysate at 40 µg

    Secondary
    HRP-conjugated goat anti-mouse polyclonal IgG at 1/2000 dilution

    Developed using the ECL technique.

    Exposure time: 20 seconds


    Blocked with 5% Milk for 2 hours at 22°C

    This image was generated using the ascites version of the product.

    See Abreview

  • Frozen sections of human stomach tissue stained for Hsp90 using ab13492 at 1/100 dilution in immunohistochemical analysis.

    This image was generated using the ascites version of the product.

  • ab13492 staining Hsp90 in Human testis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a TRIS-EDTA Buffer. Samples were incubated with primary antibody (1/500) for 30 minutes at 20°C. A HRP-conjugated Goat anti-rabbit/mouse IgG polyclonal was used as the secondary antibody.

    This image was generated using the ascites version of the product.

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab13492 (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 (ab7481) / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab13492, 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 image was generated using the ascites version of the product.

  • ab13492 staining Hsp90 in Human spleen tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Samples were incubated with primary antibody (10 ug/ml).

    This image was generated using the ascites version of the product.

  • All lanes : Anti-Hsp90 antibody [AC88] (ab13492) at 1/1000 dilution

    Lane 1 : Hsp90 native human protein
    Lane 2 : Hsp90 beta reombinant human protein
    Lane 3 : Hsp90 alpha reombinant human protein
    Lane 4 : Cell lysates prepared from heat shocked Hela cells
    Lane 5 : Cell lysates prepared from heat shocked 3T3 cells
    Lane 6 : Cell lysates prepared from heat shocked PC-12 cells
    Lane 7 : Cell lysates prepared from heat shocked CHO-K1 cells
    Lane 8 : Cell lysates prepared from heat shocked Rat-2 cells


    This image was generated using the ascites version of the product.

  • ICC/IF image of ab13492 stained HepG2 cells (ab7900). The cells were 4% PFA 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 (ab13492, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) used at a 1/250 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.

    This image was generated using the ascites version of the product.

  • ab13492 Immunoprecipitate OF hSP 90 in human AGS whole cell lysate. 200µg of cell lysate was incubated with primary antibody (1/250 in RIPA buffer) and matrix (Protein A/G) for 16 hours at 4°C.
    For western blotting anti-mouse HRP (ab6728) (1/1000) was used.

    This image was generated using the ascites version of the product.

    See Abreview

References

This product has been referenced in:
  • Dong L  et al. HSP90 interacts with HMGCR and promotes the progression of hepatocellular carcinoma. Mol Med Rep 19:524-532 (2019). Read more (PubMed: 30483734) »
  • Eckersley-Maslin M  et al. Dppa2 and Dppa4 directly regulate the Dux-driven zygotic transcriptional program. Genes Dev 33:194-208 (2019). Read more (PubMed: 30692203) »
See all 51 Publications for this product

Customer reviews and Q&As

1-8 of 8 Q&A

Answer



Ich kann bestätigen, dass wir diesen Antikörper in humaner Milz getestet haben.

Ein Kunde hat einen Abreview eingereicht der ein sehr schönes Färbungsmuster in Magen zeigt.

In der Literatur werden übereinstimmed außerdem humane Testis als positive beschrieben.

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Question
Answer

Thank you for contacting us.
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I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.
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The credit note ID is for your reference only and does not automatically guarantee the credit.
I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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Answer

Thank you for contacting us.

The immunogen should be related to the following:

http://www.uniprot.org/uniprot/Q8LLI5.

Also please note that this antibody will react with native, alpha and beta Hsp90.

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

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Question
Answer

Thank you for contacting us. As I mentioned in our converstaion, we do not have lysates of heat-shocked cells, which would be the most convincing controls, combined with lysates of untreated cells. We have lysates of HeLa treated with a variety of agents but I am not sure if any are known to upregulate HSP90 in this cell line. We have data from a customer for another HSP90 antibody (ab13494) which shows a strong signal in western blots from HEK293 cells. We have a lysate of this cell line as ab7902: Click here (or use the following: https://www.abcam.com/index.html?datasheet=7902). Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting Abcam regarding ab13492. I am sorry that your customer has been experiencing difficulties with this antibody in WB.  I have reviewed the protocol information provided and would like to make some protocol suggestions that will improve the customer's results with this antibody. Regarding the sample type, I would recommend that a positive control is included in the analysis.  Expression is notably higher in pituitary gland, brain, adrenal gland, and testis.  Also, a heat shocked sample of a human cell line such as HeLa cells would be suitable as a positive control. Regarding the protocol, I would recommend shortening the block time to just 1 hr at room temperature and increasing the incubation time with the primary antibody to overnight at 4oC.  If the protein is not abudantly present in lung, a longer incubation time will allow more time for sufficient antibody to bind.  I hope this information is helpful.  Please do not hesitate to contact us if you have any additional questions.  

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Answer

Thank you for contacting us. Unfortunately, none of the antibodies you mentioned (ab91259, ab83036, ab13492) have been tested in zebrafish so far. However, we have another HSP90 antibody that was tested in zebrafish: ab13494 (https://www.abcam.com/Hsp90-antibody-16F1-ab13494.html) - it would be guaranteed for this species as well as the listed tested applications. For Aha1, we do not have an antibody tested in this species. But if you please could send me the protein sequence for zebrafish, I'd be happy to check homology and to see whether any of the Aha1 antibodies can be used for this species. If the homology is about 85% or higher, you can use our testing discount program. For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works: The testing discount program is for customers who like to use an antibody/protein on an untested species/application. You would purchase the antibody at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody at the full price (100%) of the antibody you have tested. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 113383 DESCRIPTION OF THE PROBLEM no band SAMPLE cell lysate or purified Hsp90 PRIMARY ANTIBODY Hsp90 antibody (AC88), 1/500 or 1/1000 dilution, in 10 mM Tris, pH:7,5; 150 mM NaCl and 3% BSA, for 1 hrs, wash: 20 mM Tris, pH:7,5; 500 mM NaCl, 0,2% Triton, 2*10 min, then 10 mM Tris, pH:7,5; 150 mM NaCl for 10 min. DETECTION METHOD Chromogenic method, with NBT/BCIP POSITIVE AND NEGATIVE CONTROLS USED positive was used: purified Hsp90 (Sigma) ANTIBODY STORAGE CONDITIONS 4 0C,then -20 0C SAMPLE PREPARATION 0,06 M Tris, 2 % SDS, 5 % glycerol, ph:6,8; 1*10-6 cell/ 500 ul cell lysate, cell lysate were boiled for 5 min, at 95 0C, then centrifugated and the 20 ul was loaded to SDS page. AMOUNT OF PROTEIN LOADED 20 ul ELECTROPHORESIS/GEL CONDITIONS 10 % SDS PAGE, reducing gel, stucking: 110 V, running 180 V TRANSFER AND BLOCKING CONDITIONS Transfer: 2h, 350 mA, 100 V Wash: 10 mM Tris, pH:7,5; 150 mM NaCl, 10 min Block: above buffer+ 3 % BSA, 1 hr Wash: 20 mM Tris, pH:7,5; 500 mM NaCl, 0,2% Triton, 2*10 min SECONDARY ANTIBODY Alkaline phoshatase conjugate anti mause IgG, 1/30000 dilution, in 10 mM Tris, pH:7,5; 150 mM NaCl and 3% BSA, for 1 hrs, wash: 20 mM Tris, pH:7,5; 500 mM NaCl, 0,2% Triton, 2*10 min, then 10 mM Tris, pH:7,5; 150 mM NaCl for 10 min. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? cell lysis ADDITIONAL NOTES When we received, we stored at 4 0C for one day, then it was immidiatly used in western blot.

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Answer

Thank you for your enquiry and for completing our technical questionnaire so comprehensively. I am sorry to hear that you have been having difficulties with this antibody. I was interested in you sample preparation. In our standard, recommended western blot protocol (http://ops.abcam.com/index.html?pageconfig=view_protocol&pid=161) we advise using protease inhibitors in the sample preparation buffers. The absence of bands could be attributable to protein degradation. For Hsp90 I would recommend that you use a RIPA buffer preparation. If you would like details of this preparation please do not hesitate to contact me. I was also interested in your antibody buffer conditions. We advise that you use TBST (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.05%, Tween 20) or PBST for the incubation (supplemented with 5% BSA or non-fat Milk) and the washes. I had a little concern that the Triton conditions that you have been using were not favouring the binding of this antibody. We also recommend the detection of immunoblotting using ECL or the more sensitive ECL+. This will dramatically improve the sensitivity of your analysis. I would also recommend the use of a 7.5% SDS-PAGE gel given the molecular weight of the protein (90KDa) as detected by western analysis. Can you also tell me whether you consider what the mass of protein you have been loading is? It may be useful to load more protein into your blot e.g. >20ug. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Question

BATCH NUMBER 113455 ORDER NUMBER 14/6/05? DESCRIPTION OF THE PROBLEM Lost signal. Antibody worked great at first (received 14/6/05), but after about 3 months no signal could be obtained. New lot (#127755) received 20/9/05 shows no signal either. SAMPLE Whole cell lysate of SK-Br-3 cells. PRIMARY ANTIBODY The dilutions tested were 1:500, 1:1000 and 1:5000. The membranes were incubated with primary antibodies in Tris-buffered saline (TBS) with 0.1 % (v/v) Tween 20 and 1 % fat-free dry-milk overnight at 4?C. Washed 3 x 10 min. in Tris-buffered saline (TBS) with 0.1 % (v/v) Tween 20 at room temp. before blocking as before. DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED SK-Br-3 cells used for positive control. ANTIBODY STORAGE CONDITIONS 4?C SAMPLE PREPARATION Cells were lysed in SDS/PAGE sample buffer [10 mM Tris/HCl (pH 6.8) / 5 mM EDTA / 50 mM NaF / 30 mM sodium pyrophosphate / 2 % (w/v) SDS / 1 mM Na3VO4 / 1 mM PMSF] on ice for 10 min. Then 4 % (v/v) glycerol, 4 % (v/v) b-mercaptoethanol and 0.005 % (w/v) Bromophenol Blue were added to the lysates. The lysates were incubated at 95?C for 10 min. AMOUNT OF PROTEIN LOADED 20 ul of lysate (sufficient for strong signal). ELECTROPHORESIS/GEL CONDITIONS SDS/PAGE 10 % gel. TRANSFER AND BLOCKING CONDITIONS Transferred to Nitrocellulose membrane at 100 V for 1 hour in standard transfer buffer. Blocking solution: Tris-buffered saline [TBS; 10 mM Tris/HCl (pH 7.4) / 137 mM NaCl] with 0.1 % (v/v) Tween 20 and 5 % fat-free dry-milk. Blocked 30 min. at room temp. before primary and secondary antibody incubation. SECONDARY ANTIBODY Jackson Peroxidase-conjugated Donkey Anti-Mouse IgG (H+L) (#715-035-150) diluted 1:5000 in Tris-buffered saline (TBS) with 0.1 % (v/v) Tween 20 and 1 % fat-free dry-milk for 1 h at room temp. Washed 3 x 10 min. in Tris-buffered saline (TBS) with 0.1 % (v/v) Tween 20 at room temp. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? None.

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Answer

Thank you for your enquiry. May I also thank you for taking the time to fill out our technical questionnaire so comprehensively. I was very interested to hear that you have had such a deterioration in the quality of your results. I could understand you having difficulties with a new LOT as you mention given that these represent different antisera preparations and may require subtly different optimisation conditions. However, for an antibody to decline in quality from the same LOT, I think the problem is possibly attributable to the sample or the storage conditions of the sera. Please can you tell me whether you aliquotted the sera upon arrival and have stored it at -20oC or -80oC?. Could it be possible that the expression of Hsp90 has declined to the point of non-expression. Have you tried to detect the transcript of Hsp90 in your samples recently. This would be in keeping with your findings of a decline in quality and failure of your new LOT. Have you tried an alternative "positive control" such as various cultured cell lines. Could it be possible that your breast cancer cell lysates have deteriorated by protease digestion? You mention that you were using NaF, but perhaps a protease inhibitor cocktail would add confidence to the lysate preparation. Have you determined the integrity of your lysates using an alternative antiserum targeted against the same or different constitutively expressed protein? Could there be anything that has "expired" in the lab that was used to make up your buffers? I look forward to hearing from you.

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