Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-GRP94 antibody [EPR22847-50] (ab238126)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR22847-50] to GRP94
  • Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IP
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-GRP94 antibody [EPR22847-50]
    See all GRP94 primary antibodies

  • Description

    Rabbit monoclonal [EPR22847-50] to GRP94

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IPmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human GRP94 aa 100-200. The exact sequence is proprietary.
    Database link: P14625

  • Positive control

    • WB: Human kidney and stomach tissue lysates; mouse brain and placenta tissue lysates; rat brain, placenta and stomach tissue lysates; HeLa, MCF7, HEK293T, MEF, PC-12, C6 and NIH/3T3 whole cell lysates. IHC-P: Human kidney tissue; mouse and rat colon tissue. ICC/IF: HeLa and NIH/3T3 cells. Flow Cyt: HeLa cells. IP: HeLa whole cell lysate.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

Properties

Applications

Our Abpromise guarantee covers the use of ab238126 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
WB 1/1000. Detects a band of approximately 94 kDa (predicted molecular weight: 94 kDa).
IHC-P 1/5000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF 1/1000.
Flow Cyt 1/600.
IP 1/30.

Target

  • Function

    Molecular chaperone that functions in the processing and transport of secreted proteins. Functions in endoplasmic reticulum associated degradation (ERAD). Has ATPase activity.

  • Sequence similarities

    Belongs to the heat shock protein 90 family.

  • Cellular localization

    Endoplasmic reticulum lumen. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • 94 kDa glucose regulated protein antibody
    • 94 kDa glucose-regulated protein antibody
    • ECGP antibody
    • Endoplasmin antibody
    • Endothelial cell (HBMEC) glycoprotein antibody
    • ENPL_HUMAN antibody
    • Glucose regulated protein 94kDa antibody
    • gp96 antibody
    • gp96 homolog antibody
    • GRP 94 antibody
    • GRP-94 antibody
    • Heat shock protein 90 kDa beta member 1 antibody
    • heat shock protein 90kDa beta (Grp94), member 1 antibody
    • Heat shock protein, 90 kDa, beta, 1 antibody
    • HSP90B1 antibody
    • Stress inducible tumor rejection antigen GP96 antibody
    • TRA1 antibody
    • tumor rejection antigen (gp96) 1 antibody
    • Tumor rejection antigen 1 antibody
    • Tumor rejection antigen gp96 antibody
    • Tumor rejection antigen-1 (gp96) antibody
    see all

Images

  • Western blot - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Western blot - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    All lanes : Anti-GRP94 antibody [EPR22847-50] (ab238126) at 1/1000 dilution

    Lane 1 : Wild-type HEK293T cell lysate
    Lane 2 : HSP90B1 knockout HEK293T cell lysate
    Lane 3 : HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 94 kDa
    Observed band size: 94 kDa



    Lanes 1-3: Merged signal (red and green). Green - ab238126 observed at 94 kDa. Red - loading control, ab8245 observed at 37 kDa.

    ab238126 Anti-GRP94 antibody [EPR22847-50] was shown to specifically react with GRP94 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266313 (knockout cell lysate ab257254) was used. Wild-type and GRP94 knockout samples were subjected to SDS-PAGE. ab238126 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

     

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP94 antibody [EPR22847-50] (ab238126)

    Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling GRP94 with ab238126 at 1/5000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on rat colon (PMID: 23572575) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

    The section was incubated with ab238126 for 15 mins at RT.

    The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP94 antibody [EPR22847-50] (ab238126)

    Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling GRP94 with ab238126 at 1/5000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on mouse colon (PMID: 23572575) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

    The section was incubated with ab238126 for 15 mins at RT.

    The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP94 antibody [EPR22847-50] (ab238126)

    Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling GRP94 with ab238126 at 1/5000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human kidney (PMID: 20520781) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

    The section was incubated with ab238126 for 15 mins at RT.

    The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

  • Immunoprecipitation - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Immunoprecipitation - Anti-GRP94 antibody [EPR22847-50] (ab238126)

    GRP94 was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab238126 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab238126 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.

    Lane 1: HeLa whole cell lysate 10 μg (Input).
    Lane 2: ab238126 IP in HeLa whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab238126 in HeLa whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.
    Exposure time: 3 seconds.

  • Flow Cytometry - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Flow Cytometry - Anti-GRP94 antibody [EPR22847-50] (ab238126)

    Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling GRP94 with ab238126 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue).

    Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

  • Western blot - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Western blot - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    All lanes : Anti-GRP94 antibody [EPR22847-50] (ab238126) at 1/1000 dilution

    Lane 1 : Mouse brain tissue lysate at 20 µg
    Lane 2 : Mouse placenta tissue lysate at 20 µg
    Lane 3 : Mouse stomach tissue lysate at 20 µg
    Lane 4 : Rat brain tissue lysate at 20 µg
    Lane 5 : Rat placenta tissue lysate at 20 µg
    Lane 6 : Rat stomach tissue lysate at 20 µg
    Lane 7 : C6 (rat glial tumor glial cell line) whole cell lysate at 20 µg
    Lane 8 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 10 µg

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 94 kDa
    Observed band size: 94 kDa



    Blocking and dilution buffer: 5% NFDM/TBST.

    Exposure times.

    Lane 1: 26 seconds;  Lanes 2-5: 10 seconds; Lane 6: 92 seconds; Lanes 7-8: 6 seconds.

    The molecular weight observed is consistent with what has been described in the literature (PMID: 26858453, 29949743).

  • Western blot - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Western blot - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    All lanes : Anti-GRP94 antibody [EPR22847-50] (ab238126) at 1/1000 dilution

    Lane 1 : Human brain tissue lysate at 20 µg
    Lane 2 : Human kidney tissue lysate at 20 µg
    Lane 3 : Human stomach tissue lysate at 20 µg
    Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
    Lane 5 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg
    Lane 6 : MEF (Mouse embryonic fibroblast (immortalized) cell line) whole cell lysate at 20 µg
    Lane 7 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 94 kDa
    Observed band size: 94 kDa



    Blocking and dilution buffer: 5% NFDM/TBST.

    Exposure times.

    Lanes 1 & 2: 10 seconds; Lane 3: 37 seconds; Lanes 4-6: 10 seconds; Lane 7: 26 seconds.

    The molecular weight observed is consistent with what has been described in the literature (PMID: 26858453, 29949743).

     

  • Immunocytochemistry/ Immunofluorescence - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Immunocytochemistry/ Immunofluorescence - Anti-GRP94 antibody [EPR22847-50] (ab238126)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling GRP94 with ab238126 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000  dilution (green). Confocal image showing endoplasmic reticulum staining in NIH/3T3 cell line. The nuclear counterstain is DAPI (blue). The endoplasmic reticulum is stained with Anti-KDEL antibody [10C3] (ab12223), followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) secondary antibody (red).

    Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000  dilution.

     

  • Immunocytochemistry/ Immunofluorescence - Anti-GRP94 antibody [EPR22847-50] (ab238126)
    Immunocytochemistry/ Immunofluorescence - Anti-GRP94 antibody [EPR22847-50] (ab238126)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling GRP94 with ab238126 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000  dilution (green). Confocal image showing endoplasmic reticulum staining in HeLa cell line. The nuclear counter stain is DAPI (blue). The endoplasmic reticulum is stained with Anti-KDEL antibody [10C3] (ab12223), followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) secondary antibody (red).

    Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000  dilution.

     

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (0)

Publishing research using ab238126? Please let us know so that we can cite the reference in this datasheet.

ab238126 has not yet been referenced specifically in any publications.

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