Anti-GRP94 antibody [EPR22847-50]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GRP94 antibody [EPR22847-50] (AB238126)

Intracellular 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.

• IP

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Immunoprecipitation - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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.

All lanes:

Immunoprecipitation - Anti-GRP94 antibody [EPR22847-50] (ab238126)

Predicted band size: 92 kDa

Observed band size: 94 kDa

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• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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.

• WB

Unknown

Western blot - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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).

All lanes:

Western blot - 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:

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: 92 kDa

Observed band size: 94 kDa

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• WB

Lab

Western blot - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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.

All lanes:

Western blot - Anti-GRP94 antibody [EPR22847-50] (ab238126) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

HSP90B1 knockout HEK293T cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Predicted band size: 92 kDa

Observed band size: 94 kDa

false

• WB

Lab

Western blot - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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.

All lanes:

Western blot - Anti-GRP94 antibody [EPR22847-50] (ab238126) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

HSP90B1 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human HSP90B1 (GRP94) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-hsp90b1-grp94-knockout-hek-293t-cell-line-ab266313'>ab266313</a>)

Lane 3:

HeLa cell lysate at 20 µg

Predicted band size: 92 kDa

Observed band size: 94 kDa

false

• WB

Unknown

Western blot - Anti-GRP94 antibody [EPR22847-50] (AB238126)

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:

Western blot - 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:

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: 92 kDa

Observed band size: 94 kDa

false