Anti-Emerin antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Emerin antibody (AB40688)

ICC/IF image of ab40688 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab40688, 1μg/ml) for 1h at room temperature. 1%BSA / 10% normal serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

• IP

Unknown

Immunoprecipitation - Anti-Emerin antibody (AB40688)

Emerin was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Emerin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab40688.
Secondary : Clean-Blot IP Detection Reagent (HRP) at 1/500 dilution.
Band : 33kDa; Emerin;non specific bands - 52 and 60kDa : We are unsure as to the identity of this extra band.

All lanes:

Immunoprecipitation - Anti-Emerin antibody (ab40688)

Predicted band size: 29 kDa

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• WB

Lab

Western blot - Anti-Emerin antibody (AB40688)

Lanes 1-2 : Merged signal (red and green). Green - ab40688 observed at 35 kDa. Red - loading control ab8245 observed at 37 kDa.

ab40688 Anti-Emerin antibody was shown to specifically react with Emerin in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266336 (knockout cell lysate ab257423) was used. Wild-type and Emerin knockout samples were subjected to SDS-PAGE. ab40688 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Emerin antibody (ab40688) at 1 µg/mL

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

EMD knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human EMD (Emerin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-emd-emerin-knockout-hek-293t-cell-line-ab266336'>ab266336</a>)

Predicted band size: 29 kDa

Observed band size: 35 kDa

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• WB

Lab

Western blot - Anti-Emerin antibody (AB40688)

Lanes 1 - 3 : Merged signal (red and green). Green - ab40688 observed at 35 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab40688 was shown to recognize Emerin in wild-type HAP1 cells as signal was lost at the expected MW in Emerin knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Emerin knockout samples were subjected to SDS-PAGE. ab40688 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Emerin antibody (ab40688) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Emerin knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Predicted band size: 29 kDa

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• WB

Project2870****

Western blot - Anti-Emerin antibody (AB40688)

All lanes:

Western blot - Anti-Emerin antibody (ab40688) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg

Lane 3:

Western blot - Jurkat whole cell lysate (<a href='/en-us/products/cell-lysates/jurkat-whole-cell-lysate-ab7899'>ab7899</a>) at 10 µg

Lane 4:

Jurkat nuclear extract lysate (<a href='/en-us/products/unavailable/jurkat-nuclear-extract-lysate-ab14844'>ab14844</a>) at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 29 kDa

Observed band size: 33 kDa

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