Overview

  • Product name
    Anti-KDEL antibody [10C3]
    See all KDEL primary antibodies
  • Description
    Mouse monoclonal [10C3] to KDEL
  • Specificity
    This antibody specifically recognizes proteins containing the SEKDEL sequence in mammals, birds, insects and plants. It recognises GPR78 and GPR94 with particular prominence. N.B Recent customer feedback has provided evidence that this antibody more specifically recognises the SEKDEL sequence rather than simply the KDEL sequence.
  • Tested applications
    Suitable for: IHC-P, IHC-Fr, ICC/IF, WB, ELISA, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Chinese hamster
    Predicted to work with: Bird, Plants, a wide range of other species, Mammal
  • Immunogen

    Peptide sequence SEKDEL corresponding to amino acids 649-654 of rat Grp78 conjugated to KLH.

  • Positive control
    • HeLa cell lysate

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer
    Preservative: None
    Constituents: 50% Glycerol, PBS, 0.1mM PMSF. pH 7.2
  • Concentration information loading...
  • Purity
    Protein G purified
  • Clonality
    Monoclonal
  • Clone number
    10C3
  • Isotype
    IgG2a
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab12223 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 Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
WB Use a concentration of 4 µg/ml. Predicted molecular weight: 78 , 94 kDa.
ELISA Use a concentration of 5 - 10 µg/ml.
IP Use at an assay dependent concentration.
Flow Cyt Use 0.5µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

Target

  • Relevance
    The sequence Lys-Asp-Glu-Leu (KDEL) or a closely related sequence, is present at the carboxy-terminus of soluble endoplasmic reticulum (ER) resident proteins and some membrane proteins. 78 and 94 kDa glucose regulated proteins (GRP 78) and GRP 94 respectively and protein disulfide isomerase (PDI) all share the C-terminal KDEL sequence. The presence of carboxy-terminal KDEL appears to be necessary for ER retention and appears to be sufficient to reduce the secretion of proteins from the ER. This retention is reported to be mediated by a KDEL receptor.
  • Cellular localization
    Endoplasmic reticulum
  • Database links
  • Alternative names
    • KDEL antibody
    • Lys Asp Glu Leu antibody

Anti-KDEL antibody [10C3] images

  • ICC/IF image of ab12223 stained HeLa cells. 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 (ab12223, 10µg/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.



  • Predicted band size : 78 , 94 kDa
    Three constructs were generated (denoted protein one to three). Proteins 1 and 2 end in SEKDEL as the C-terminal sequence. Protein two is an original construct which ends in GGKDEL. These were expressed and separated on a 12% SDS-PAGE, blotted to PVDF and blocked with 4% milk in TBS/0.1% tween. The replicates were then treated with either a specific antibody for the expressed protein (B), the KDEL antibody - ER Marker (ab2898) or KDEL antibody [10C3] - ER Marker (ab12223). A secondary antibody conjugated to alkaline phosphatase was used to detect the primary. The Coomassie stained gel indicates the location and expression levels of the three proteins. The specific antibody confirms the bands as the proteins of interest. KDEL antibody [10C3] - ER Marker (ab12223) recognizes proteins ending in the SEKDEL sequence and does not recognize the GGKDEL sequence as detected by KDEL antibody - ER Marker (ab2898).
  • All lanes : Anti-KDEL antibody [10C3] (ab12223) at 5 µg/ml

    Lane 1 : Purified human prekallikrein
    Lane 2 : Normal human plasma
    Lane 3 : Normal human plasma


    Predicted band size : 78 , 94 kDa

    Bands of 85kDa and 88kDa are detected.

  • ab12223 staining KDEL in mouse liver tissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with formaldehyde and permeabilized with 0.2% Triton X-100 before blocking with 2% BSA for 30 minutes at 200C. The sample was incubated with primary antibody (1/200) for 9 hours at 40C. An Alexa Fluor® 555-conjugated Goat polyclonal to mouse IgG was used as secondary antibody at dilution at 1/200. DAPI was used to stain the cell nuclei (blue).

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab12223 (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 (ab12223, 0.5µg/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 IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • ab12223 at 1/250 staining a mouse macropahge cell line by ICC/IF. The cells were paraformaldehyde fixed and blocked with BSA prior to incubation with the antibody for 24 hours. An Alexa-Fluor ® 488 conjugated donkey anti-mouse antibody was used as the secondary.

    See Abreview

  • ab12223 staining human HeLa cells by ICC/IF. Cells were PFA fixed and permeabilized in 0.1% Triton X-100 for 10 minutes prior to blocking in 3% BSA for 15 minutes at room temperature. The primary antibody was diluted 1/50 and incubated with sample for 1 hour. A Alexa Fluor® 568 goat polyclonal to mouse IgG was used as the secondary, diluted 1/1000.

    See Abreview

  • ab12223 staining KDEL in Mouse kidney tissue by Immunohistochemistry (Formalin/ PFA-fixed paraffin-embedded tissue sections). The sections were Formaldehyde-fixed and subjected to Heat-mediated antigen retrieval in citric acid (pH 6.0) and permeabilized with 0.2% Triton X-100 prior to blocking with 5% BSA for 2 hours at 25°C. The primary antibody was diluted 1/250 in PBS and incubated with the sample for 9 hours at 4°C. An Alexa Fluor® 594-conjugated Goat anti-Mouse IgG polyclonal was used as the secondary antibody, diluted 1/500. A DAPI counterstain was used for nuclei.

    See Abreview

References for Anti-KDEL antibody [10C3] (ab12223)

This product has been referenced in:
  • Dobroff AS  et al. Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes. Proc Natl Acad Sci U S A N/A:N/A (2016). Flow Cyt ; Human . Read more (PubMed: 27791177) »
  • Kasetti RB  et al. Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork. Invest Ophthalmol Vis Sci 57:6058-6069 (2016). Mouse . Read more (PubMed: 27820874) »

See all 31 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Kidney)
Specification
Kidney
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer pH 6.0
Permeabilization
Yes - 0.2% Triton X-100
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Abcam user community

Verified customer

Submitted Nov 25 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Tissue lysate - whole (293 FT cels)
Loading amount
50 µg
Specification
293 FT cels
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
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Abcam user community

Verified customer

Submitted Sep 01 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (Testis)
Specification
Testis
Fixative
Formaldehyde
Permeabilization
Yes - 0.1% Triton X-100
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 16 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Bone marrow cells)
Specification
Bone marrow cells
Fixative
Formaldehyde
Permeabilization
Yes - 0.1% Triton X-100
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Verified customer

Submitted May 20 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (293FT)
Specification
293FT
Fixative
Formaldehyde
Permeabilization
No
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Abcam user community

Verified customer

Submitted Jan 14 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Spleen)
Specification
Spleen
Fixative
Acetone
Permeabilization
No
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
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Verified customer

Submitted Jan 04 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (Liver)
Loading amount
100 µg
Specification
Liver
Gel Running Conditions
Non-reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Verified customer

Submitted Nov 18 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Liver)
Specification
Liver
Fixative
Formaldehyde
Permeabilization
Yes - 0.2% Triton X-100
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 20°C
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Abcam user community

Verified customer

Submitted Nov 10 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (huh-7 cells)
Loading amount
15000 cells
Specification
huh-7 cells
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

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Verified customer

Submitted Jul 27 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100 (10 mins)
Blocking step
BSA as blocking agent for 15 minute(s) · Concentration: 3% · Temperature: RT°C
Username

Dr. Dan Tattersall

Verified customer

Submitted Nov 05 2008

1-10 of 17 Abreviews or Q&A

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