Anti-ERK1 antibody [EP4967]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ERK1 antibody [EP4967] (AB109282)

Intracellular Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling ERK1 with purified ab109282 at 1/30 dilution (red). The secondary antibody was Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ERK1 antibody [EP4967] (AB109282)

Flow cytometry overlay histogram showing wild-type Hap1 (green line) and MAPK3 knockout Hap1 stained with ab109282 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab109282) (1x 106 in 100μl at 0.2 μg/ml (1/11400)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, MAPK3 knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 antibody [EP4967] (AB109282)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human melanoma tissue labelling ERK1 with purified ab109282 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 antibody [EP4967] (AB109282)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colonic adenocarcinoma tissue labelling ERK1 with unpurified ab109282 at 1/100 dilution.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ERK1 antibody [EP4967] (AB109282)

Immunocytochemistry/Immunofluorescence analysis of Jurkat (human acute T cell leukemia) cells labelling ERK1 with purified ab109282 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• IP

Lab

Immunoprecipitation - Anti-ERK1 antibody [EP4967] (AB109282)

ERK1 was immunoprecipitated from Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate with ab109282 at 1/50 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab109282 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-ERK1 antibody [EP4967] (ab109282) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate (Input) at 10 µg

Lane 2:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate (+)

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab109282 in Jurkat whole cell lysate (-)

Secondary

All lanes:

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

Observed band size: 44 kDa

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Exposure time: 15s

• IP

Lab

Immunoprecipitation - Anti-ERK1 antibody [EP4967] (AB109282)

ERK1 was immunoprecipitated from NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab109282 at 1/50 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab109282 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-ERK1 antibody [EP4967] (ab109282) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 2:

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

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab109282 in NIH/3T3 whole cell lysate (-)

Secondary

All lanes:

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

Observed band size: 44 kDa

false

Exposure time: 15s

• WB

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Western blot - Anti-ERK1 antibody [EP4967] (AB109282)

Lanes 1, 3 and 5 : Wild-type HAP1 cell lysate (20 μg)
Lanes 2, 4 and 6 : ERK1 knockout HAP1 cell lysate (20 μg)
Lanes 1 and 2 : Green signal from target - ab109282 observed at 42 kDa
Lanes 3 and 4 : Red signal from loading control - ab8226 observed at 42 kDa
Lanes 5 and 6 : Merged (red and green) signal
ab109282 was shown to specifically react with ERK1 when ERK1 knockout samples were used.
Wild-type and ERK1 knockout samples were subjected to SDS-PAGE. ab109282 and ab8226 (loading control to beta actin) were both diluted 1/1000 and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ERK1 antibody [EP4967] (ab109282)

Predicted band size: 43 kDa

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• WB

Lab

Western blot - Anti-ERK1 antibody [EP4967] (AB109282)

Lanes 1- 2 : Merged signal (red and green). Green - ab109282 observed at 43 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab109282 was shown to react with ERK1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266519 (knockout cell lysate ab257099) was used. Wild-type HEK-293T and MAPK3 knockout HEK-293T 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. ab109282 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-ERK1 antibody [EP4967] (ab109282) at 1/1000 dilution

Lane 1:

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

Lane 2:

MAPK3 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human MAPK3 (ERK1) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-mapk3-erk1-knockout-hek-293t-cell-line-ab266519'>ab266519</a>)

Predicted band size: 43 kDa

Observed band size: 43 kDa

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• WB

Lab

Western blot - Anti-ERK1 antibody [EP4967] (AB109282)

Predicted band size : 43 kDa

Lanes 1, 3 and 5 : Wild-type HAP1 cell lysate (20 μg)
Lanes 2, 4 and 6 : ERK1 knockout HAP1 cell lysate (20 μg)
Lanes 1 and 2 : Green signal from target
Lanes 3 and 4 : Red signal from loading control
Lanes 5 and 6 : Merged (red and green) signal

Red - loading control, ab8226 observed at 42 kDa or ab8245, observed at 37 kDa

This western blot image is a comparison between ab109282 and a competitor's top cited rabbit polyclonal antibody.

All lanes:

Western blot - Anti-ERK1 antibody [EP4967] (ab109282)

Predicted band size: 43 kDa

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• WB

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Western blot - Anti-ERK1 antibody [EP4967] (AB109282)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-ERK1 antibody [EP4967] (ab109282) at 1/10000 dilution

Lane 1:

Jurkat cell lysate at 20 µg

Lane 2:

A375 cell lysate at 20 µg

Lane 3:

HUVEC 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 1/10000 dilution

Predicted band size: 114 kDa,15 kDa,18 kDa,21 kDa,34 kDa,43 kDa,55 kDa,68 kDa,83 kDa

Observed band size: 15 kDa,21 kDa,23 kDa,90 kDa

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• WB

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Western blot - Anti-ERK1 antibody [EP4967] (AB109282)

All lanes:

Western blot - Anti-ERK1 antibody [EP4967] (ab109282) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

A375 cell lysate at 10 µg

Lane 3:

A431 cell lysate at 10 µg

Lane 4:

HUVEC cell lysate at 10 µg

Predicted band size: 43 kDa

false

• WB

Unknown

Western blot - Anti-ERK1 antibody [EP4967] (AB109282)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-ERK1 antibody [EP4967] (ab109282) at 1/10000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Lane 3:

NIH/3T3 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 1/10000 dilution

Predicted band size: 43 kDa

false