Western blot - GRP78 BiP antibody (ab21685)

All lanes : Anti-GRP78 BiP antibody (ab21685) at 1 µg/ml

Lane 1 : CHO-K1 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 whole cell lysate at 20 µg
Lane 5 : CHO-K1 whole cell lysate at 20 µg/ml with GRP78 BiP peptide (ab22410) at 1 µg
Lane 6 : Liver (Mouse) Tissue Lysate at 20 µg with GRP78 BiP peptide (ab22410) at 1 µg/ml
Lane 7 : Rat liver whole cell lysate at 20 µg with GRP78 BiP peptide (ab22410) at 1 µg/ml
Lane 8 : HeLa whole cell lysate at 20 µg with GRP78 BiP peptide (ab22410) at 1 µg/ml

Secondary
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.

Immunocytochemistry/ Immunofluorescence - GRP78 BiP antibody (ab21685)

ICC/IF image of ab21685 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab21685, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Immunocytochemistry/ Immunofluorescence - GRP78 BiP antibody (ab21685)

ICC/IF image of ab21685 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab21685, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - GRP78 BiP antibody (ab21685)

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.

Western blot - GRP78 BiP antibody (ab21685)

All lanes : Anti-GRP78 BiP antibody (ab21685) at 1 µg/ml

Lane 1 : CHO-K1 cell lysate 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
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 - GRP78 BiP antibody (ab21685)

ab21685 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).

This image was kindly supplied by Dr Vladimir Milenkovic by Abreview

Immunocytochemistry/ Immunofluorescence - GRP78 BiP antibody (ab21685)

ab21685 at a 1/500 dilution staining GRP78 BiP in Dog MDCK II cells by Immunocytochemistry/ Immunofluorescence, incubated for 16 hours at 4ºC. PFA fixed. Blocked with 5% serum for 20 minutes at 25ºC. Secondary used at a 1/500 dilution polyclonal Goat anti-rabbit conjugated to Alexa Fluor 488 (green). Nuclei were counterstained with DAPI (blue).

This image was kindly supplied by Dr Vladimir Milenkovic by Abreview

Immunocytochemistry/ Immunofluorescence - GRP78 BiP antibody (ab21685)

ICC/IF image of ab21685 stained Hela cells. The cells were 100% methanol fixed (5 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 (ab21685, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) 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

All lanes : Anti-GRP78 BiP antibody (ab21685) at 1 µg/ml

Lane 1 : GRP78 BiP protein (Active) (ab78432) at 0.1 µg
Lane 2 : GRP78 BiP protein (Active) (ab78432) at 0.01 µg

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Exposure time : 10 seconds