重组Anti-eIF4A2抗体[EPR27347-74]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling eIF4A2 with ab307728 at 1/50 (9.92 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic and weak nuclear staining in HeLa cell line.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling eIF4A2 with ab307728 at 1/10000 (0.05 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on human testis. The section was incubated with ab307728 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling eIF4A2 with ab307728 at 1/500 dilution (0.1ug) (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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Immunohistochemical analysis of paraffin-embedded Human breast carcino tissue labeling eIF4A2 with ab307728 at 1/10000 (0.05 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on human breast carcinoma (PMID : 21219636). The section was incubated with ab307728 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IP

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Immunoprecipitation - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

eIF4A2 was immunoprecipitated from 0.35 mg HeLa whole cell lysate with ab307728 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307728 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate Lane 2 : ab307728 IP in HeLa whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307728 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 1 seconds

All lanes:

Immunoprecipitation - Anti-eIF4A2 antibody [EPR27347-74] (ab307728) at 1/30 dilution

Secondary

All lanes:

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

false

Exposure time: 1s

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling eIF4A2 with ab307728 at 1/10000 (0.05 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on rat testis. The section was incubated with ab307728 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized C6 (rat glial tumor glial cell) cells labelling eIF4A2 with ab307728 at 1/50 (9.92 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic and very weak nuclear staining in C6 cell line.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized C6 (rat glial tumor glial cell) cells labelling eIF4A2 with ab307728 at 1/500 dilution (0.1ug) (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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling eIF4A2 with ab307728 at 1/10000 (0.05 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on mouse testis. The section was incubated with ab307728 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling eIF4A2 with ab307728 at 1/500 dilution (0.1ug) (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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Raw 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling eIF4A2 with ab307728 at 1/50 (9.92 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic and weak nuclear staining in Raw 264.7 cell line.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• WB

Lab

Western blot - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Western blot : Anti-EIF4A2 antibody [EPR27347-74] (ab307728) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab307728 was shown to bind specifically to EIF4A2. A band was observed at 48 kDa in wild-type A549 cell lysates with no signal observed at this size in EIF4A2 knockout cell line. To generate this image, wild-type and EIF4A2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-eIF4A2 antibody [EPR27347-74] (ab307728) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

EIF4A2 knockout A549 cell lysate at 20 µg

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

MCF7 Membrane Prep cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 48 kDa

false

• WB

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Western blot - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Low expression : liver(PMID : 8521730). In Western blot, anti- H3 antibody (ab176842) loading control staining at 1/100000 dilution. Exposure time : 6 seconds

All lanes:

Western blot - Anti-eIF4A2 antibody [EPR27347-74] (ab307728) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 20 µg

Lane 2:

Human heart tissue lysate at 20 µg

Lane 3:

Human liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 48 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : 10 seconds

All lanes:

Western blot - Anti-eIF4A2 antibody [EPR27347-74] (ab307728) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 5:

Human testis tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 48 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The identity of the lower MW band at approximately 20 kDa is unknown. Exposure time : 15 seconds

All lanes:

Western blot - Anti-eIF4A2 antibody [EPR27347-74] (ab307728) at 1/1000 dilution

Lane 1:

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

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 3:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 4:

C2C12 (mouse myoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 48 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-eIF4A2 antibody [EPR27347-74] (AB307728)

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

This antibody does not cross-react with human EIF4A1 and human EIF4A3.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-eIF4A2 antibody [EPR27347-74] (ab307728) at 1/1000 dilution

Lane 1:

His-tagged human EIF4A1 recombinant protein at 10 ng

Lane 2:

His-tagged human EIF4A2 recombinant protein at 10 ng

Lane 3:

His-tagged human EIF4A3 recombinant protein at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 48 kDa

true

Exposure time: 48s