Anti-eIF5A 抗体 [EP526Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-eIF5A antibody [EP526Y] (AB32443)

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling eIF5A with purified ab32443 at 1/500. Cells were fixed with 4% Paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei couterstained with DAPI (blue).

Secondary Only Control : PBS was used instead of the primary antibody as the negative control

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF5A antibody [EP526Y] (AB32443)

ab32443 at a 1 : 250 dilution staining eIF5A in human adenocarcinoma of uterus using Immunohistochemistry, Paraffin Embedded Tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-eIF5A antibody [EP526Y] (AB32443)

Overlay histogram showing Jurkat cells stained with ab32443 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32443, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (0.5μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-eIF5A antibody [EP526Y] (AB32443)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized C6 (rat glial tumor glial cell) labelling eIF5A with ab32443 at 1/250 dilution (Green).

Confocal image showing Mitochondria staining in C6 cell line.

Nuclear DNA was labelled with DAPI (shown in blue).

Secondary used was ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 µg/ml).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody -Microtubule Marker (Alexa Fluor® 594) was used as a counterstain at 1/200 (2.5 µg/ml) dilution.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-eIF5A antibody [EP526Y] (AB32443)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) labelling eIF5A with ab32443 at 1/250 dilution (Green).

Confocal image showing Mitochondria staining in NIH/3T3 cell line.

Nuclear DNA was labelled with DAPI (shown in blue).

Secondary used was ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 µg/ml).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody -Microtubule Marker (Alexa Fluor® 594) was used as a counterstain at 1/200 (2.5 µg/ml) dilution.

Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF5A antibody [EP526Y] (AB32443)

Immunohistochemical analysis of paraffin-embedded Mouse pancreas labeling eIF5A with ab32443 at 1/1000 dilution (0.101 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the Mouse pancreas. The section was incubated with ab32443 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF5A antibody [EP526Y] (AB32443)

Immunohistochemical analysis of paraffin-embedded Rat pancreas labeling eIF5A with ab32443 at 1/1000 dilution (0.101 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the Rat pancreas. The section was incubated with ab32443 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF5A antibody [EP526Y] (AB32443)

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle labeling eIF5A with ab32443 at 1/1000 dilution (0.101 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : Negligible staining on the mouse cardiac muscle. The section was incubated with ab32443 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• WB

Supplier Data

Western blot - Anti-eIF5A antibody [EP526Y] (AB32443)

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

Low expression control : mouse and rat heart.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-eIF5A antibody [EP526Y] (ab32443) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate  at 20 µg

Lane 2:

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

Lane 3:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 4:

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

Lane 5:

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

Lane 6:

Mouse pancreas tissue lysate at 20 µg

Lane 7:

Mouse heart tissue lysate at 20 µg

Lane 8:

Rat pancreas tissue lysate at 20 µg

Lane 9:

Rat heart 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 1/20000 dilution

Predicted band size: 16 kDa

Observed band size: 16 kDa

false

Exposure time: 1s

• WB

Unknown

Western blot - Anti-eIF5A antibody [EP526Y] (AB32443)

All lanes:

Western blot - Anti-eIF5A antibody [EP526Y] (ab32443) at 1/10000 dilution

All lanes:

Jurkat cell lysate

Predicted band size: 16 kDa

Observed band size: 18 kDa

false

• WB

Lab

Western blot - Anti-eIF5A antibody [EP526Y] (AB32443)

Blocking buffer : 5% NFDM/TBST

This antibody shows low affinity in recognizing eIF5A2 recombinant protein.

All lanes:

Western blot - Anti-eIF5A antibody [EP526Y] (ab32443) at 1/2000 dilution

Lane 1:

Western blot - Recombinant Human eIF5A protein (<a href='/products/proteins-peptides/recombinant-human-eif5a-protein-ab87457'>ab87457</a>) at 0.01 µg

Lane 2:

Western blot - Recombinant Human eIF5A2 protein (<a href='/products/proteins-peptides/recombinant-human-eif5a2-protein-ab99140'>ab99140</a>) at 0.01 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 1/20000 dilution

Predicted band size: 16 kDa

Observed band size: 15 kDa,20 kDa

true

Exposure time: 20s