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 (Human umbilical vein endothelial cell) 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 seconds

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

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.

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.

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 with Mouse GRP78 BiP peptide (ab22410) at 1 µg
Lane 6 : Liver (Mouse) Tissue Lysate at 20 µg with Mouse GRP78 BiP peptide (ab22410) at 1 µg/ml
Lane 7 : Rat liver whole cell lysate at 20 µg with Mouse GRP78 BiP peptide (ab22410) at 1 µg/ml
Lane 8 : HeLa whole cell lysate at 20 µg with Mouse 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.

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.

IHC image of GRP78 BiP staining in human liver carcinoma FFPE section, performed on a Bond^TM 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.

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

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 analysis of MDA-MB-435S 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.

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