Anti-GRP78 BiP antibody

• IHC-P

Unknown

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 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP78 BiP antibody (AB21685)

Immunohistochemical analysis of formalin fixed paraffin embedded human liver carcinoma labelling GRP78 BiP with ab21685 at a concentration of 0.4µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab21685 Anti-GRP78 BiP antibody was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• ICC/IF

AbReview58303****

Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (AB21685)

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.

This image is courtesy of an anonymous abreview.

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (AB21685)

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.

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/

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (AB21685)

ab21685 staining GRP78 BiP in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab21685 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Project

Western blot - Anti-GRP78 BiP antibody (AB21685)

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.

All lanes:

Western blot - 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 (<a href='/en-us/products/unavailable/mouse-grp78-bip-peptide-ab22410'>ab22410</a>)

Lane 6:

Liver (Mouse) Tissue Lysate at 20 µg with Mouse GRP78 BiP peptide (<a href='/en-us/products/unavailable/mouse-grp78-bip-peptide-ab22410'>ab22410</a>)

Lane 7:

Rat liver whole cell lysate at 20 µg with Mouse GRP78 BiP peptide (<a href='/en-us/products/unavailable/mouse-grp78-bip-peptide-ab22410'>ab22410</a>)

Lane 8:

HeLa whole cell lysate at 20 µg with Mouse GRP78 BiP peptide (<a href='/en-us/products/unavailable/mouse-grp78-bip-peptide-ab22410'>ab22410</a>)

Secondary

All lanes:

Goat anti Rabbit IgG at 1/10000 dilution

Predicted band size: 72 kDa

Observed band size: 100 kDa,75 kDa

false

• WB

Lab

Western blot - Anti-GRP78 BiP antibody (AB21685)

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

All lanes:

Western blot - Anti-GRP78 BiP antibody (ab21685) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

HeLa treated with 2.5 μg/ml tunicamycin for 24h whole cell lysates at 20 µg

Lane 3:

HUVEC (Human umbilical vein endothelial cell) whole cell lysates at 20 µg

Lane 4:

HUEVC (Human umbilical vein endothelial cell) treated with 10 μg/ml tunicamycin for 48h whole cell lysates at 20 µg

Lane 5:

Raw 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg

Lane 6:

Raw 264.7 treated with 5 μg/ml tunicamycin for 18h whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 72 kDa

Observed band size: 78 kDa

false

Exposure time: 3s

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-GRP78 BiP antibody (AB21685)

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.

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/

• WB

Ap

Western blot - Anti-GRP78 BiP antibody (AB21685)

All lanes:

Western blot - 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

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 72 kDa

Observed band size: 100 kDa,78 kDa

true

Exposure time: 1min

• WB

Unknown

Western blot - Anti-GRP78 BiP antibody (AB21685)

All lanes:

Western blot - Anti-GRP78 BiP antibody (ab21685) at 1 µg/mL

Lane 1:

Western blot - Recombinant human GRP78 BiP protein (Active) (<a href='/en-us/products/proteins-peptides/recombinant-human-grp78-bip-protein-active-ab78432'>ab78432</a>) at 0.1 µg

Lane 2:

Western blot - Recombinant human GRP78 BiP protein (Active) (<a href='/en-us/products/proteins-peptides/recombinant-human-grp78-bip-protein-active-ab78432'>ab78432</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 72 kDa

true

Exposure time: 10s