Anti-Flotillin 2/ESA antibody [EPR26975-52]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Flotillin 2/ESA antibody [EPR26975-52] (AB307422)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue lABeling Flotillin 2/ESA with ab307422 at 1/1000 (0.535 ug/ml) followed by a ready to use RABbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human tonsil. The section was incubated with ab307422 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-Flotillin 2/ESA antibody [EPR26975-52] (AB307422)

Immunohistochemical analysis of paraffin-embedded Human hepatocellular tissue lABeling Flotillin 2/ESA with ab307422 at 1/1000 (0.535 ug/ml) followed by a ready to use RABbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human hepatocellular carcinoma (PMID : 28560058). The section was incubated with ab307422 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-Flotillin 2/ESA antibody [EPR26975-52] (AB307422)

Immunohistochemical analysis of paraffin-embedded Human breast carcino tissue lABeling Flotillin 2/ESA with ab307422 at 1/1000 (0.535 ug/ml) followed by a ready to use RABbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human breast carcinoma (PMID : 22869152). The section was incubated with ab307422 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-Flotillin 2/ESA antibody [EPR26975-52] (AB307422)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HEK293 tissue lABeling Flotillin 2/ESA with ab307422 at 1/1000 (0.535 ug/ml) followed by a ready to use RABbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on (A) wild-type HEK293T cell pellet, no staining on (B) FLOT2 knockout HEK293T cell pellet. The section was incubated with ab307422 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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Flotillin 2/ESA antibody [EPR26975-52] (AB307422)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type 293T (human embryonic kidney epithelial cell, Right) / Flotillin 2/ESA knockout 293T (Left) cells labelling Flotillin 2/ESA with ab307422 at 1/50 dilution (1ug) (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 Flotillin 2/ESA knockout 293T cells (ab266847).

• IP

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Immunoprecipitation - Anti-Flotillin 2/ESA antibody [EPR26975-52] (AB307422)

Flotillin 2/ESA was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) whole cell lysate 10 ug with ab307422 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307422 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate 10 ug Lane 2 : ab307422 IP in HEK-293T whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307422 in HEK-293T whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 24 seconds

All lanes:

Immunoprecipitation - Anti-Flotillin 2/ESA antibody [EPR26975-52] (ab307422) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney epithelial cell) whole cell lysate 10 μg

Lane 2:

HEK-293T whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab307422 in HEK-293T whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 47 kDa

false

Exposure time: 24s

• WB

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Western blot - Anti-Flotillin 2/ESA antibody [EPR26975-52] (AB307422)

Blocking and diluting buffer and concentration : Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBSThe samples were run on a Bis-Tris gel. Performed under reducing conditions. False colour image of Western blot : Anti-Flotillin 2/ESA antibody [EPR26975-52] (ab307422) 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, ab307422 was shown to bind specifically to Flotillin 2/ESA. A band was observed at 47 kDa in wild-type HEK-293T cell lysates whereas no signal observed at this size in Flotillin 2/ESA knockout cell line ab266847 (knockout cell lysate ab257442). . To generate this image, wild-type and Flotillin 2/ESA knockout HEK-293T cell lysates were analyzed. First, samples were run on a 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 ЎгC. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) at 1/10000 dilution and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution. Exposure time :

All lanes:

Western blot - Anti-Flotillin 2/ESA antibody [EPR26975-52] (ab307422) at 1/1000 dilution

Lane 1:

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

Lane 2:

Flotillin 2/ESA knockout HEK-293T whole cell lysate 20 μg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate 20 μg

Secondary

Lanes 1 - 3:

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 1/10000 dilution

Lanes 1, 2 and 4:

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

Observed band size: 47 kDa

false

• WB

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Western blot - Anti-Flotillin 2/ESA antibody [EPR26975-52] (AB307422)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time :

All lanes:

Western blot - Anti-Flotillin 2/ESA antibody [EPR26975-52] (ab307422) at 1/1000 dilution

Lane 1:

Human breast tissue lysate 20 μg

Lane 2:

Human skin tissue lysate 20 μg

Lane 3:

Human lung tissue lysate 20 μg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 47 kDa

false

Exposure time: 114s