Anti-ERK1 antibody [EP4967] - BSA and Azide free

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 antibody [EP4967] - BSA and Azide free (AB214169)

This IHC data was generated using the same anti-EKR1 antibody clone, EP4967, in a different buffer formulation (cat# 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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ERK1 antibody [EP4967] - BSA and Azide free (AB214169)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ERK1 antibody [EP4967] - BSA and Azide free (AB214169)

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 Fluor^® 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109282).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ERK1 antibody [EP4967] - BSA and Azide free (AB214169)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109282).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 antibody [EP4967] - BSA and Azide free (AB214169)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109282).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-ERK1 antibody [EP4967] - BSA and Azide free (AB214169)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] (<a href='/en-us/products/primary-antibodies/erk1-antibody-ep4967-ab109282'>ab109282</a>) 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 <a href='/en-us/products/primary-antibodies/erk1-antibody-ep4967-ab109282'>ab109282</a> 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

false

Exposure time: 15s

• IP

Lab

Immunoprecipitation - Anti-ERK1 antibody [EP4967] - BSA and Azide free (AB214169)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] (<a href='/en-us/products/primary-antibodies/erk1-antibody-ep4967-ab109282'>ab109282</a>) 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 <a href='/en-us/products/primary-antibodies/erk1-antibody-ep4967-ab109282'>ab109282</a> 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] - BSA and Azide free (AB214169)

This WB data was generated using the same anti-EKR1 antibody clone, EP4967, in a different buffer formulation (cat# 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] (<a href='/en-us/products/primary-antibodies/erk1-antibody-ep4967-ab109282'>ab109282</a>)

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-ERK1 antibody [EP4967] - BSA and Azide free (AB214169)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/erk1-antibody-ep4967-ab109282'>ab109282</a>) 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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