Overview

  • Product name

    Anti-Hsp90 alpha antibody [2G5.G3]
    See all Hsp90 alpha primary antibodies
  • Description

    Mouse monoclonal [2G5.G3] to Hsp90 alpha
  • Host species

    Mouse
  • Specificity

    ab79849 is Hsp90 alpha specific (>96% alpha specific by ELISA).
  • Tested applications

    Suitable for: Flow Cyt, IHC-Fr, IHC-P, ELISA, WB, IP, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Human Hsp90 alpha

  • Positive control

    • WB: HAP1 and HeLa whole cell lysate. IHC-P: Human testis tissue. ICC/IF: HaCaT cells. Flow Cyt: HeLa cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab79849 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
Flow Cyt Use 1µg for 106 cells.

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

IHC-Fr Use at an assay dependent concentration.
IHC-P Use a concentration of 0.5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ELISA Use at an assay dependent concentration.
WB 1/2000. Predicted molecular weight: 85 kDa.
IP Use at an assay dependent concentration.
ICC/IF 1/100.

Target

  • Function

    Molecular chaperone. Has ATPase activity.
  • Sequence similarities

    Belongs to the heat shock protein 90 family.
  • Post-translational
    modifications

    ISGylated.
  • Cellular localization

    Cytoplasm. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt
  • Database links

  • Alternative names

    • EL52 antibody
    • epididymis luminal secretory protein 52 antibody
    • Heat shock 86 kDa antibody
    • heat shock 90kD protein 1, alpha antibody
    • Heat shock 90kD protein 1, alpha like 4 antibody
    • heat shock 90kD protein, alpha-like 4 antibody
    • Heat shock 90kDa protein 1 alpha antibody
    • Heat shock protein 90kDa alpha (cytosolic) class A member 1 antibody
    • Heat shock protein HSP 90-alpha antibody
    • HS90A_HUMAN antibody
    • HSP 86 antibody
    • HSP86 antibody
    • Hsp89 antibody
    • HSP89A antibody
    • Hsp90 antibody
    • HSP90A antibody
    • HSP90AA1 antibody
    • HSP90ALPHA antibody
    • HSP90N antibody
    • HSPC1 antibody
    • HSPCA antibody
    • HSPCAL1 antibody
    • HSPCAL4 antibody
    • HSPN antibody
    • LAP 2 antibody
    • LAP2 antibody
    • lipopolysaccharide-associated protein 2 antibody
    • LPS-associated protein 2 antibody
    • Renal carcinoma antigen NY-REN-38 antibody
    see all

Images

  • Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
    Lane 2: Hsp90 alpha knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)

    Lanes 1 - 3: Merged signal (red and green). Green - ab79849 observed at 90 kDa. Red - loading control, ab181602, observed at 37 kDa.

    ab79849 was shown to specifically react with Hsp90 alpha in wild-type HAP1 cells as signal was lost in Hsp90 alpha knockout cells. Wild-type and Hsp90 alpha knockout samples were subjected to SDS-PAGE. Ab79849 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

     

  • IHC image of Hsp90 alpha staining in Human Testis formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab79849, 0.5 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

     

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ab79849 at 1/100 dilution staining Hsp90 alpha in human keratinocyte cell line HaCaT by Immunocytochemistry/ immunofluorescence. A Fluorophore conjugated goat anti mouse was used as secondary at 1/50 dilution.

  • Overlay histogram showing HeLa cells stained with ab79849 (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 / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab79849, 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.

References

This product has been referenced in:

  • Marrugal Á  et al. Impact of Heat Shock Protein 90 Inhibition on the Proteomic Profile of Lung Adenocarcinoma as Measured by Two-Dimensional Electrophoresis Coupled with Mass Spectrometry. Cells 8:N/A (2019). Read more (PubMed: 31370342) »
  • Watanabe M  et al. CD30 Induces Heat Shock Protein 90 and Signal Integration in Classic Hodgkin Lymphoma Cells. Am J Pathol 187:163-175 (2017). Read more (PubMed: 27870927) »
See all 10 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Question
Answer

Thank you for yoru recent telephone enquiry.

I ampleased to let you know I now have the ELISA testing protocol for ab79849 -Hsp90 alpha antibodyfrom the originator. Thank you for your patience.
I have copied this below:

Purified hsp90alpha (1 μg) was coated on each well of a Maxisorp C96 titer plate (Nalge Nunc Int, Rochester, NY, USA) were incubated with various concentrations of purified mAbs in 0.1 mL of the blocking buffer (0.17 M H3BO4, pH 8.5, containing 0.12 M NaCl, 0.05% [vol/vol] Tween 20, 1 mM ethylenediaminetetraacetic acid, and 0.05% [wt/vol] NaN3 and 0.25% [wt/vol] bovine serum albumin) for 2 hours at room temperature.

After a 1-hour incubation with the anti-mouse Ig(G+A+M) and IgE conjugated to alkaline phosphatase (1/4000 dilution) and washing, the alkaline phosphatase activity was determined by measuring the absorbance at 655 nm after a 1-hour incubation with Bluephos microwell phosphatase substrate at 30°C.


Please note, this protocol would be a guideline only, and may require some individual optimization for individual experiments. I can also suggest to check the datasheet for ab79849 for further information. For example, it is tested and guaranteed for Mouse, Rat, Human. Which species will you be using?

https://www.abcam.com/index.html?datasheet=79849 (or use the following:
https://www.abcam.com/index.html?datasheet=79849).

I hope this will be helpful. If you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for your reply. I will forward your request to 51AB, who will be in contact with you to process your refund request. 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 your reply with this information. It seems that the lower band is certainly isoform 1 of HSP90 and it is likely that the higher band is isoform2. I am not an expert in this field and could not say for sure if both isoforms are expressed in your samples. If you are dissatisfied with these results, I am happy to replace the antibody, however it is possible you will see a similar result. Please do not hesitate to contact me if you have any additional questions.

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Answer

Thank you for contacting Abcam regarding ab79849. I am sorry that you have been experiencing difficulties with this antibody in WB. I have reviewed the data provided and would like to point out that there is a second isoform of this protein that is predicted to be 98kDa and may be represented by this band. SwissProt: P07900 What are the blocking conditions that you tested? Also, have you tested other primary antibody dilutions? How long do you incubate with the primary antibody? I hope this information is helpful. I look forward to your reply so that I may assist you further. Please do not hesitate to contact me with additional questions.

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Answer

Than you for your inquiry and I am sorry to hear that you are experiencing some trouble with ab53497 and ab79849. Are you running any isotype controls with these experiments? I just want to make sure that the pull down with the HBP21 antibody is genuine. Have you tried to do a regular IP and not a co-IP with ab53497 and ab79849? These are the IP references we know of with the HSP beta antibody, ab53497. Barent R. L. (1998) Mol. Endocrinol. 12: 342-354 11. Lo. M.A. (1998) EMBO J. 17: 6879-6887. What kind of lysis buffer are you using? A key factor in maintaining complex formation throughout the steps required for co-IP is the lysis and wash buffers. Many protein interactions will remain intact after lysis using standard non-denaturing lysis buffers. Buffers with low ionic strength (i.e., <120mM NaCl) that contain non-ionic detergents (NP-40 and Triton X-100) are less likely to disrupt protein-protein interactions; however, testing may be required to determine the best buffer formulation for a specific protein complex of interest. Additionally, lysing cells by sonication or vortexing the lysate or bead-bound immune complexes during the wash steps should be avoided to prevent the disruption of the protein-protein interaction(s) of the target complex. And while centrifugation is a standard method to separate the precipitated complexes from the remaining lysate and during wash steps, the samples should be handled gently to prevent the loss of bound complex proteins. An advanced technique to strengthen protein-protein interactions is by crosslinking the binding partners. Using this approach, all proteins within the active distance of the specific reagent in a cell lysate are covalently crosslinked, and the target protein can then be immunoprecipitated along with the other proteins in the complex without the risk of losing binding partners. Co-IPs can be tricky due to the nature of the interaction of the proteins, nonspecific binding to IP components and antibody contamination that may mask detection. We haven't tested ab53497 and ab79849 by co-IP so it's difficult to know if this may be the issue. Could you try the protocol recommendations or run a regular IP with the antibody? That could show that it is an issue with the protein binding more than the antibodies.

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Application
Western blot
Sample
Human Cell lysate - whole cell (primary cancer-associated fibroblasts (prostate))
Loading amount
30 µg
Specification
primary cancer-associated fibroblasts (prostate)
Gel Running Conditions
Reduced Denaturing (Invitrogen's NuPAGE 4-12%)
Blocking step
BSA as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Dr. Alexander Henke

Verified customer

Submitted Aug 02 2011

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