All lanes : Anti-GRP94 antibody [EPR22847-50] (ab238126) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : HSP90B1 knockout HEK293T cell lysate
Lane 3 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 94 kDa
Observed band size: 94 kDa

Lanes 1-3: Merged signal (red and green). Green - ab238126 observed at 94 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab238126 Anti-GRP94 antibody [EPR22847-50] was shown to specifically react with GRP94 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266313 (knockout cell lysate ab257254) was used. Wild-type and GRP94 knockout samples were subjected to SDS-PAGE. ab238126 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling GRP94 with ab238126 at 1/5000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on rat colon (PMID: 23572575) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab238126 for 15 mins at RT.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling GRP94 with ab238126 at 1/5000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on mouse colon (PMID: 23572575) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab238126 for 15 mins at RT.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling GRP94 with ab238126 at 1/5000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human kidney (PMID: 20520781) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab238126 for 15 mins at RT.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

GRP94 was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab238126 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab238126 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 μg (Input).
Lane 2: ab238126 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab238126 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling GRP94 with ab238126 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-GRP94 antibody [EPR22847-50] (ab238126) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate at 20 µg
Lane 2 : Mouse placenta tissue lysate at 20 µg
Lane 3 : Mouse stomach tissue lysate at 20 µg
Lane 4 : Rat brain tissue lysate at 20 µg
Lane 5 : Rat placenta tissue lysate at 20 µg
Lane 6 : Rat stomach tissue lysate at 20 µg
Lane 7 : C6 (rat glial tumor glial cell line) whole cell lysate at 20 µg
Lane 8 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 94 kDa
Observed band size: 94 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 26 seconds;  Lanes 2-5: 10 seconds; Lane 6: 92 seconds; Lanes 7-8: 6 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 26858453, 29949743).

All lanes : Anti-GRP94 antibody [EPR22847-50] (ab238126) at 1/1000 dilution

Lane 1 : Human brain tissue lysate at 20 µg
Lane 2 : Human kidney tissue lysate at 20 µg
Lane 3 : Human stomach tissue lysate at 20 µg
Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 5 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg
Lane 6 : MEF (Mouse embryonic fibroblast (immortalized) cell line) whole cell lysate at 20 µg
Lane 7 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 94 kDa
Observed band size: 94 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lanes 1 & 2: 10 seconds; Lane 3: 37 seconds; Lanes 4-6: 10 seconds; Lane 7: 26 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 26858453, 29949743).

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling GRP94 with ab238126 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000  dilution (green). Confocal image showing endoplasmic reticulum staining in NIH/3T3 cell line. The nuclear counterstain is DAPI (blue). The endoplasmic reticulum is stained with Anti-KDEL antibody [10C3] (ab12223), followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) secondary antibody (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000  dilution.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling GRP94 with ab238126 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000  dilution (green). Confocal image showing endoplasmic reticulum staining in HeLa cell line. The nuclear counter stain is DAPI (blue). The endoplasmic reticulum is stained with Anti-KDEL antibody [10C3] (ab12223), followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) secondary antibody (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000  dilution.