Recombinant 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% TBS. The 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 °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. 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.

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

Exposure times:

Lane 1: 114 seconds
Lane 2: 92 seconds
Lane 3: 15 seconds

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Flotillin 2/ESA with ab307422 at 1/1000 dilution (0.535 µg/ml) followed by 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 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.

Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling Flotillin 2/ESA with ab307422 at 1/1000 dilution (0.535 µg/ml) followed by 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 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.

Immunohistochemical analysis of paraffin-embedded cell pellets labeling Flotillin 2/ESA with ab307422 at 1/1000 dilution (0.535 µg/ml) followed by 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 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.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling Flotillin 2/ESA with ab307422 at 1/1000 dilution (0.535 µg/ml) followed by 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 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 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 labeling Flotillin 2/ESA with ab307422 at 1/50 dilution (1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled 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).

Flotillin 2/ESA was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) whole cell lysate 10 µg with ab307422 at 1/30 dilution (2µg 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 whole cell lysate 10 µg

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.