The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
1/1000 - 1/2000. Detects a band of approximately 78 kDa (predicted molecular weight: 72 kDa).
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
FunctionProbably 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 diseaseAutoantigen in rheumatoid arthritis.
Sequence similaritiesBelongs to the heat shock protein 70 family.
Cellular localizationEndoplasmic reticulum lumen. Melanosome. Cytoplasm. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
IHC image of GRP78 BiP staining in Human breast fibroadenoma 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 ab109659, 1µg/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.
ICC/IF image of ab109659 stained HeLa cells. The cells were 4% formaldehyde 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 (ab109659, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 Goat anti-Rabbit IgG (H+L) 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.
Western blot - GRP78 BiP antibody (ab109659)
All lanes : Anti-GRP78 BiP antibody (ab109659) at 1 µg/ml
Lane 1 : Recombinant Human GRP78 BiP at 0.1 µg Lane 2 : SKBR3 lysate (Human) at 7.5 µg Lane 3 : MDCK lysate (Dog) at 7.5 µg Lane 4 : MEF lysate (Mouse) at 7.5 µg