Anti-GRP78 BiP antibody (ab21685)
Rabbit polyclonal GRP78 BiP antibody. Validated in WB, IP, IHC, EM, ICC/IF and tested in Mouse, Rat, Dog, Human, Pig, African green monkey, Chinese hamster. Cited in 206 publication(s).
- Datasheet
- References (284)
- Protocols
Overview
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Product name
Anti-GRP78 BiP antibody
See all GRP78 BiP primary antibodies -
Description
Rabbit polyclonal to GRP78 BiP -
Host species
Rabbit -
Tested applications
Suitable for: IHC-FoFr, IP, ICC/IF, WB, Electron Microscopy, IHC-Pmore details -
Species reactivity
Reacts with: Mouse, Rat, Dog, Human, Pig, African green monkey, Chinese hamster -
Immunogen
Synthetic peptide corresponding to Mouse GRP78 BiP aa 600 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
(Peptide available asab22410) -
Positive control
- WB: Mouse and Rat liver tissue lysates; Raw 264.7 whole cell lysates, CHO-K1, HUVEC, and HeLa whole cell lysates; Recombinant human GRP78 BiP protein. ICC-IF: Hela cells; HEK-293 cells transiently transfected with wild-type murine IGSF1-2-Myc/His; COS-7 cells; Mouse retinal pigment epithelium primary cells; MDCK II cells; MDA-MB-435 cells. IHC-P: Human liver carcinoma tissue; Panc1 tumor taken out of Balb/C nude mice.
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General notes
Stimulation may be required to allow detection of the target protein due to low levels of endogenous expression in some samples. Please see images below for recommended treatment conditions and positive controls.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help. -
Concentration information loading... -
Purity
Immunogen affinity purified -
Clonality
Polyclonal -
Isotype
IgG -
Research areas
Associated products
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Compatible Secondaries
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Immunizing Peptide (Blocking)
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Isotype control
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Recombinant Protein
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Related Products
Applications
Our Abpromise guarantee covers the use of ab21685 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-FoFr | Use at an assay dependent concentration. | |
| IP | Use a concentration of 2 µg/ml. | |
| ICC/IF | Use a concentration of 1 - 5 µg/ml. We recommend using Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody. |
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| WB | Use a concentration of 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 78 kDa).Can be blocked with Mouse GRP78 BiP peptide (ab22410). | |
| Electron Microscopy | 1/250. | |
| IHC-P | Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. |
Target
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Function
Probably plays a role in facilitating the assembly of multimeric protein complexes inside the endoplasmic reticulum. Involved in the correct folding of proteins and degradation of misfolded proteins via its interaction with DNAJC10, probably to facilitate the release of DNAJC10 from its substrate. -
Involvement in disease
Autoantigen in rheumatoid arthritis. -
Sequence similarities
Belongs to the heat shock protein 70 family. -
Cellular localization
Endoplasmic reticulum lumen. Melanosome. Cytoplasm. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. - Information by UniProt
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Database links
- Entrez Gene: 100689305 Chinese hamster
- Entrez Gene: 480726 Dog
- Entrez Gene: 3309 Human
- Entrez Gene: 14828 Mouse
- Entrez Gene: 407060 Pig
- Entrez Gene: 25617 Rat
- Omim: 138120 Human
- SwissProt: P11021 Human
see all -
Alternative names
- 78 kDa glucose regulated protein antibody
- 78 kDa glucose-regulated protein antibody
- AL022860 antibody
see all
Images
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Western blot - Anti-GRP78 BiP antibody (ab21685)All lanes : Anti-GRP78 BiP antibody (ab21685) at 1/1000 dilution
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : HeLa treated with 2.5 µg/ml tunicamycin for 24h whole cell lysates
Lane 3 : HUVEC (Human umbilical vein endothelial cell) whole cell lysates
Lane 4 : HUEVC treated with 10 µg/ml tunicamycin for 48h whole cell lysates
Lane 5 : Raw 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
Lane 6 : Raw 264.7 treated with 5 µg/ml tunicamycin for 18h whole cell lysates
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 78 kDa
Observed band size: 78 kDa
Exposure time: 3 secondsBlocking/Diluting buffer and concentration: 5% NFDM/TBST
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Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (ab21685)Image from Wang Y et al., PLoS One. 2017;12(7):e0180731. Fig 3.; doi: 10.1371/journal.pone.0180731. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/HEK-293 cells were cultured on coverslips and transiently transfected with wild-type murine IGSF1-2-Myc/His, premeabilized, and processed for double-label immunofluorescence with the Myc antibody (green) and an antibody against GRP78 BiP (red). The overlay is shown in yellow. Nuclei were stained with DAPI. Images were captured by confocal microscopy. Scale bar, 10 μm.
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Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (ab21685)Image from Friebe S et al., PLoS One. 2015;10(3):e0119864. Fig 3.; doi: 10.1371/journal.pone.0119864. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/Localization of TEM8 and CMG2 glycosylation mutants.
A) Immunofluorescence of transiently transfected HeLa cells. Cells were transfected for 48h with the respective cDNAs. Cells were fixed, permeabilized and stained for TEM8-HA, endogenous BiP and Hoechst. Scalebars represent 10 μm.
B) Immunofluorescence of transiently transfected HeLa cells. Cells were transfected for 48h with the respective cDNAs. Cells were fixed, permeabilized and stained for CMG2-V5, endogenous BiP and Hoechst. Scalebars represent 10 μm.
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Western blot - Anti-GRP78 BiP antibody (ab21685)All lanes : Anti-GRP78 BiP antibody (ab21685) at 1 µg/ml
Lane 1 : CHO-K1 (chinese hamster ovary cell line) whole cell lysate at 20 µg
Lane 2 : Liver (Mouse) Tissue Lysate at 20 µg
Lane 3 : Rat liver whole cell lysate at 20 µg
Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 5 : CHO-K1 whole cell lysate at 20 µg/ml withMouse GRP78 BiP peptide (ab22410) at 1 µg
Lane 6 : Liver (Mouse) Tissue Lysate at 20 µg withMouse GRP78 BiP peptide (ab22410) at 1 µg/ml
Lane 7 : Rat liver whole cell lysate at 20 µg withMouse GRP78 BiP peptide (ab22410) at 1 µg/ml
Lane 8 : HeLa whole cell lysate at 20 µg withMouse GRP78 BiP peptide (ab22410) at 1 µg/ml
Secondary
All lanes : Goat anti Rabbit IgG at 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 78 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?
Additional bands at: 100 kDa. We are unsure as to the identity of these extra bands.ab21685 recognises a band of ~ 75 kDa in CHO, mouse liver, rat liver and HeLa whole cell lysates, corresponding to GRP78 BiP. This band is quenched by the addition of the immunizing peptide, ab22410.
ab21685 also detects a 100 kDa band in Western Blot. We are unsure of the identity of this protein.
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Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (ab21685)ab21685 stained in Hela cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab21685 at 1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) (pseudo-colored red) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP78 BiP antibody (ab21685)IHC image of GRP78 BiP staining in human liver carcinoma FFPE section, performed on a BondTM 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 ab21685, 1µg/ml, for 8 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. -
Western blot - Anti-GRP78 BiP antibody (ab21685)All lanes : Anti-GRP78 BiP antibody (ab21685) at 1 µg/ml
Lane 1 : CHO-K1 (chinese hamster ovary cell line) Whole Cell Lysate
Lane 2 : Liver (Mouse) Tissue Lysate at 10 µg
Lane 3 : Liver (Rat) Tissue Lysate at 10 µg
Lane 4 : HeLa (human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
Secondary
Lanes 1 & 4 : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Lanes 2-3 : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 78 kDa
Observed band size: 78 kDa
Additional bands at: 100 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 1 minute -
Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (ab21685)This image is courtesy of an anonymous Abreviewab21685 staining GRP78 BiP in African Green Monkey COS-7 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 3% BSA for 1 hour at 23°C. Samples were incubated with primary antibody (1/1000 in PBS-BSA) for 1 hour at 23°C. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP78 BiP antibody (ab21685)This image is courtesy of an anonymous abreview.ab21685 staining Panc1 tumor taken out of Balb/C nude mice sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). The tissue was fixed with formaldehyde and a heat mediated antigen retrival step was performed with citric acid pH 6. Staining with ab21685 at 1 µg/ml for 18 hours at 4°C. An undiluted goat anti-rabbit polyclonal HRP conjugated secondary antibody was used.
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Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (ab21685)This image was kindly supplied by Dr Vladimir Milenkovic by Abreviewab21685 at a 1/500 dilution staining GRP78 BiP in mouse retinal pigment epithelium primary cells by Immunocytochemistry/ Immunofluorescence, incubated for 16 hours at 4°C. PFA fixed. Blocked with 5% serum for 20 minutes at 25°C. Secondary used at a 1/500 dilution polyclonal Goat anti-rabbit conjugated to Alexa Fluor 488 (green). Nuclei were counterstained with DAPI (blue). -
Western blot - Anti-GRP78 BiP antibody (ab21685)All lanes : Anti-GRP78 BiP antibody (ab21685) at 1 µg/ml
Lane 1 :Recombinant human GRP78 BiP protein (Active) (ab78432) at 0.1 µg
Lane 2 :Recombinant human GRP78 BiP protein (Active) (ab78432) at 0.01 µg
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 78 kDa
Exposure time: 10 seconds -
Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (ab21685)This image was kindly supplied by Dr Vladimir Milenkovic by Abreviewab21685 at a 1/500 dilution staining GRP78 BiP in Dog MDCK II cells by Immunocytochemistry/ Immunofluorescence, incubated for 16 hours at 4°C. PFA fixed. Blocked with 5% serum for 20 minutes at 25°C. Secondary used at a 1/500 dilution polyclonal Goat anti-rabbit conjugated to Alexa Fluor 488 (green). Nuclei were counterstained with DAPI (blue). -
Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (ab21685)This image is courtesy of an anonymous abreview.Immunocytochemistry/ Immunofluorescence analysis of MDA-MB-435 tumor cell line cells labeling GRP78 BiP with ab21685 at 1/400 dilution. Cells were fixed in formaldehyde and permeabilized with 0.25% Triton-X100 in PBS for 10 minutes. Blocking was done with 1%BSA for 1 hour at 20°C; followed by staining with ab21685 at 1/400 for 18 hours. Undiluted ab150077, a Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) secondary antibody was used. DAPI was used to counterstain.
Protocols
References
This product has been referenced in:
- Jiang L et al. Inhibition of microRNA-103 attenuates inflammation and endoplasmic reticulum stress in atherosclerosis through disrupting the PTEN-mediated MAPK signaling. J Cell Physiol 235:380-393 (2020). Read more (PubMed: 31232476) »
- Gill JF et al. Peroxisome proliferator-activated receptor ? coactivator 1a regulates mitochondrial calcium homeostasis, sarcoplasmic reticulum stress, and cell death to mitigate skeletal muscle aging. Aging Cell 18:e12993 (2019). Read more (PubMed: 31290266) »
