Anti-Egr1 antibody [EPR15916]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Egr1 antibody [EPR15916] (AB194357)

Confocal image showing nuclear and weakly cytoplasmic staining on Hela cells.

Immunocytometry/Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma ) cells labeling Egr1 with ab194357 at 1/200 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution. The cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000) using ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary. Nuclei counterstained with DAPI (blue).

For negative control 1, ab194357 (1 : 200) was used, followed by (ab150120) anti-mouse secondary antibody at 1/1000 dilution. For negative control 2, (ab7291) mouse primary antibody (1/1000) was used followed by (ab150077) anti-rabbit secondary antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Egr1 antibody [EPR15916] (AB194357)

Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue labeling Egr1 with ab194357 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Counter stained with hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-Egr1 antibody [EPR15916] (AB194357)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Egr1 antibody [EPR15916] (ab194357) at 1/50000 dilution

All lanes:

293T treated with 20% FBS at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 57 kDa

Observed band size: 80 kDa

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• WB

Lab

Western blot - Anti-Egr1 antibody [EPR15916] (AB194357)

False colour image of Western blot : Anti-Egr1 antibody [EPR15916] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab194357 was shown to bind specifically to Egr1. A band was observed at 73 kDa in wild-type HeLa cell lysates with no signal observed at this size in EGR1 CRISPR-Cas9 edited cell line ab274929 (CRISPR-Cas9 edited cell lysate ab274987). The band observed in the CRISPR-Cas9 edited lysate lane below 73 kDa is likely to represent a truncated form of Egr1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and EGR1 CRISPR-Cas9 edited HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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.

Lanes 1 - 4:

Western blot - Anti-Egr1 antibody [EPR15916] (ab194357) at 1/10000 dilution

Lanes 1 - 4:

Western blot - Anti-Egr1 antibody [EPR15916] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/egr1-antibody-epr15916-bsa-and-azide-free-ab232448'>ab232448</a>) at 1/10000 dilution

Lane 1:

Wild-type HeLa Vehicle Control TNF-a (0ng/mL, 1h) cell lysate at 20 µg

Lane 2:

Wild-type HeLa TNF-a (20ng/mL, 1h) cell lysate at 20 µg

Lane 3:

Western blot - Human EGR1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-egr1-knockout-hela-cell-line-ab274929'>ab274929</a>)

Lane 3:

Western blot - Human EGR1 knockout HeLa cell lysate (ab274987)

Lane 3:

EGR1 knockout HeLa Vehicle Control TNF-a (0ng/mL, 1h) cell lysate at 20 µg

Lane 4:

EGR1 knockout HeLa TNF-a (20ng/mL, 1h) cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 73 kDa

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