Anti-Hsp 90 alpha antibody [EPR27270-11]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type Wild-type 293T (human embryonic kidney epithelial cell, Right) / Hsp90 alpha knockout 293T(Left) cells labelling Hsp90 alpha with ab303516 at 1/50 dilution (1ug) (Red) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Positive staining on 293T cells (ab255449), while no staining on Hsp90 alpha knockout 293T cells (ab266591).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue labeling Hsp90 alpha with ab303516 at 1/1000 (0.462 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/Dab (ab209101). Positive staining on human lung carcinoma. The section was incubated with ab303516 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/Dab (ab209101).Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Immunohistochemical analysis of paraffin-embedded (A) parental HEK293T tissue labeling Hsp90 alpha with ab303516 at 1/2000 dilution (0.231 μg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining was observed on (A) parental HEK293T cell pellet, no staining is observed on (B) Hsp90 alpha KO HEK293T cell pellet. The section was incubated with ab303516 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) added without primary.

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Hsp90 alpha with ab303516 at 1/1000 (0.462 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/Dab (ab209101). Positive staining on human testis. The section was incubated with ab303516 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/Dab (ab209101).Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling Hsp90 alpha with ab303516 at 1/1000 dilution (0.462 µg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining was observed on human breast carcinoma. The section was incubated with ab303516 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at Ready to use dilution. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling Hsp90 alpha with ab303516 at 1/1000 dilution (0.462 µg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control : no staining is observed on human skeletal muscle. The section was incubated with ab303516 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling Hsp90 alpha with ab303516 at 1/1000 dilution (0.462 μg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining was observed on rat testis. The section was incubated with ab303516 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at Ready to use dilution.

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling Hsp90 alpha with ab303516 at 1/1000 dilution (0.462 μg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining was observed on mouse testis. The section was incubated with ab303516 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at Ready to use dilution.

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

• WB

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Western blot - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Blocking / Diluting buffer and concentration : 5% NFDM/TBST Negative control : skeletal muscle (PMID : 21209834)

All lanes:

Western blot - Anti-Hsp 90 alpha antibody [EPR27270-11] (ab303516) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Human skeletal muscle tissue lysate 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: 85 kDa

Observed band size: 90 kDa

false

Exposure time: 48s

• WB

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Western blot - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Blocking / Diluting buffer and concentration : 5% NFDM/TBST The samples were run on a Bis-Tris gel. Performed under reducing conditions. False colour image of Western blot : Anti-Hsp90 alpha antibody (ab303516) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab303516 was shown to bind specifically to Hsp90 alpha. A band was observed at 90 kDa in wild-type 293T cell lysates with no signal observed at this size in Hsp90 alpha knockout cell line. To generate this image, wild-type and Hsp90 alpha knockout 293T cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.

All lanes:

Western blot - Anti-Hsp 90 alpha antibody [EPR27270-11] (ab303516) at 1/1000 dilution

Lane 1:

Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Hsp90 alpha knockout 293T whole cell lysate at 20 µg

Lane 3:

Human testis tissue lysate, at 20 µg

Lane 4:

Human kidney tissue lysate at 20 µg

Lane 5:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 6:

C6 (rat glial tumor glial cell), whole cell lysate at 20 µg

Secondary

Lanes 1 - 6:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 6:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 90 kDa

false

• WB

Supplier Data

Western blot - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

Blocking / Diluting buffer and concentration : 5% NFDM/TBST Negative control : skeletal muscle (PMID : 21209834)

All lanes:

Western blot - Anti-Hsp 90 alpha antibody [EPR27270-11] (ab303516) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Human skeletal muscle tissue lysate 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: 85 kDa

Observed band size: 90 kDa

false

Exposure time: 114s

• WB

Supplier Data

Western blot - Anti-Hsp 90 alpha antibody [EPR27270-11] (AB303516)

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

All lanes:

Western blot - Anti-Hsp 90 alpha antibody [EPR27270-11] (ab303516) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Mouse testis tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

Rat testis tissue lysate 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: 85 kDa

Observed band size: 90 kDa

false

Exposure time: 114s