Anti-ERK5 antibody [EP791Y]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ERK5 antibody [EP791Y] (AB40809)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ERK5 with purified ab40809 at 1/50 dilution (10 ug/ml) (red). Cells were fixed with 80% methanol. A Goat anti rabbit IgG (Alexa Fluorr^®488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ERK5 antibody [EP791Y] (AB40809)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ERK5 with Purified ab40809 at 1 : 100 (4.8 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 μg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ERK5 antibody [EP791Y] (AB40809)

Overlay histogram showing A549 cells stained with unpurified ab40809 (red line). The cells were fixed with 80% 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 (ab40809, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IP

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Immunoprecipitation - Anti-ERK5 antibody [EP791Y] (AB40809)

ab40809 (purified) at 1 : 30 dilution (2ug) immunoprecipitating ERK5 in HeLa whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab40809 & HeLa whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab40809 in HeLa whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-ERK5 antibody [EP791Y] (ab40809)

Predicted band size: 88 kDa

Observed band size: 115 kDa

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• WB

Lab

Western blot - Anti-ERK5 antibody [EP791Y] (AB40809)

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

All lanes:

Western blot - Anti-ERK5 antibody [EP791Y] (ab40809) at 1/2000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 15 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 88 kDa

Observed band size: 115 kDa

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• WB

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Western blot - Anti-ERK5 antibody [EP791Y] (AB40809)

The predicted weight of 89 kDa is for the precursor version of human ERK5 protein. However, ab40809 detects endogenous levels of total Erk5 protein which appears around 115 kDa in SDS PAGE

All lanes:

Western blot - Anti-ERK5 antibody [EP791Y] (ab40809) at 1/5000 dilution

All lanes:

Hela cell lysate at 10 µg

Predicted band size: 88 kDa

Observed band size: 115 kDa

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• WB

Lab

Western blot - Anti-ERK5 antibody [EP791Y] (AB40809)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : MAPK7 (ERK5) knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab40809 observed at 88 kDa. Red - loading control, ab9484, observed at 37 kDa.

Unpurified ab40809 was shown to specifically react with ERK5 in wild-type HAP1 cells as signal was lost in MAPK7 (ERK5) knockout cells. Wild-type and MAPK7 (ERK5) knockout samples were subjected to SDS-PAGE. Unpurified ab40809 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ERK5 antibody [EP791Y] (ab40809)

Predicted band size: 88 kDa

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• WB

Lab

Western blot - Anti-ERK5 antibody [EP791Y] (AB40809)

Lanes 1- 2 : Merged signal (red and green). Green - ab40809 observed at 115 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab40809 was shown to react with ERK5 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265508 (knockout cell lysate ab258042) was used. Wild-type HeLa and MAPK7 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab40809 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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-ERK5 antibody [EP791Y] (ab40809) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MAPK7 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human MAPK7 (ERK5) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-mapk7-erk5-knockout-hela-cell-line-ab265508'>ab265508</a>)

Predicted band size: 88 kDa

Observed band size: 115 kDa

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• WB

Supplier Data

Western blot - Anti-ERK5 antibody [EP791Y] (AB40809)

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

In Western blot, ab323508 was shown to bind specifically to ERK5 (phospho T733). Target of interest was observed at 115 kDa in treated wild-type HeLa cell lysates (lanes -2-4) with no signal observed at this size in treated ERK5 knockout cell line (lanes 6-8) (lanes 5-8, knockout cell line ab265508 / knockout cell lysate ab258042).

Lane 1-8 on the left : untreated membrane;
Lane 1-8 on the right : membrane treated with alkaline phosphatase.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-ERK5 antibody [EP791Y] - Total protein control (ab40809) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (<a href='/en-us/products/primary-antibodies/erk5-phospho-t733-antibody-epr28258-101-ab323508'>ab323508</a>) at 1/1000 dilution

Lane 1:

Untreated parental HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Parental HeLa treated with 0.4M Sorbitol for 30min whole cell lysate at 20 µg

Lane 3:

Parental HeLa treated with 100nM Calyculin A for 30min whole cell lysate at 20 µg

Lane 4:

Parental HeLa treated with 0.4M Sorbitol and 100nM Calyculin A for 30min whole cell lysate at 20 µg

Lane 5:

Untreated ERK5 knockout HeLa whole cell lysate at 20 µg

Lane 6:

ERK5 knockout HeLa treated with 0.4M Sorbitol for 30min whole cell lysate at 20 µg

Lane 7:

ERK5 knockout HeLa treated with 100nM Calyculin A for 30min whole cell lysate at 20 µg

Lane 8:

ERK5 knockout HeLa treated with 0.4M Sorbitol and 100nM Calyculin A for 30min whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 115 kDa,36 kDa

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Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-ERK5 antibody [EP791Y] (AB40809)

Lane 1-4 on the left : untreated membrane;
Lane 1-4 on the right : membrane treated with alkaline phosphatase.

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

In Western blot, Anti-ERK5 antibody [EP791Y] - Total protein control (ab40809) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (<a href='/en-us/products/primary-antibodies/erk5-phospho-t733-antibody-epr28258-101-ab323508'>ab323508</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

NIH/3T3 treated with 0.4M Sorbitol for 30min whole cell lysate at 20 µg

Lane 3:

NIH/3T3 treated with 100nM Calyculin A for 30min whole cell lysate at 20 µg

Lane 4:

NIH/3T3 treated with 0.4M Sorbitol and 100nM Calyculin A for 30min whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 115 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-ERK5 antibody [EP791Y] (AB40809)

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

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

In Western blot, Anti-ERK5 antibody [EP791Y] - Total protein control (ab40809) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (<a href='/en-us/products/primary-antibodies/erk5-phospho-t733-antibody-epr28258-101-ab323508'>ab323508</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells co-transfected with a human ERK5 WT expression vector containing a myc-His-tag® and a rat MEK5(S311D/T315D mutation)) expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 2:

293T (human embryonic kidney epithelial cell) cells co-transfected with a human ERK5 (T733A mutation) expression vector containing a myc-His-tag® and a rat MEK5(S311D/T315D mutation)) expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 115 kDa

false

Exposure time: 70s