重组Anti-ERK1抗体[Y72] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Overlay histogram showing HeLa cells stained with ab32537 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween 20 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 (ab32537, 1/11312) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 0.01μg/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.

This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween 20 for 20 min used under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Immunocytochemistry/Immunofluorescence analysis of Jurkat cells labelling ERK1 with purified ab32537 at a dilution of 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 (1/1000) 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 (1/1000).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-MAPK3 knockout cells (red line) stained with ab32537. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab32537, 1μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. A Rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-MAPK3 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Clone Y72 (ab214168) has been successfully conjugated by Abcam. This image was generated using Anti-ERK1 antibody [Y72] (Alexa Fluor^® 647). Please refer to ab190579 for protocol details.

Overlay histogram showing HeLa cells stained with ab190579 (red line). The cells were fixed with 4% formaldehyde and then permeabilized with 0.1% PBS-Triton X-100 for 15 min.

The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab190579, 1/5000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Alexa Fluor^® 647 (ab199093) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 40 mW Red laser (640nm) and 670/14 bandpass filter.

This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

• ICC/IF

AbReview41593****

Immunocytochemistry/ Immunofluorescence - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Unpurified ab32537 staining ERK1 in HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. ab150081, a goat anti rabbit Alexa Fluor^® 488 (1/200) was used as the secondary antibody. Counterstained with DAPI. Cytoplasmic and nuclear staining shown.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

This image is courtesy of an Abreview submitted by Kirk McManus.

• Flow Cyt (Intra)

AbReview44028****

Flow Cytometry (Intracellular) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Unpurified ab32537 staining ERK1 (green) in HEK293 cells by intracellular flow cytometry. Cells were fixed with paraformaldehyde and permeabilized with 70% methanol. The sample was incubated with the primary antibody (1/40 in PBS + 0.2% BSA + 0.1% sodium azide) for 1 hour at 22°C. A phycoerythrin-conjugated goat anti-rabbit IgG (1/100) was used as the secondary antibody. Gating Strategy : Live Cells. Purple plot represents isotype control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

This image is courtesy of an anonymous Abreview

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Clone Y72 (ab214168) has been successfully conjugated by Abcam. This image was generated using Anti-ERK1 antibody [Y72] (PE). Please refer to ab210828 for protocol details.

Overlay histogram showing Jurkat cells stained with ab210828 (red line). The cells were fixed with 4% formaldehyde and then permeabilized with 90% methanol at -20°C for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab210828, 1/5000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Clone Y72 (ab214168) has been successfully conjugated by Abcam. This image was generated using Anti-ERK1 antibody [Y72] (Alexa Fluor^® 488). Please refer to ab190200 for protocol details.

Overlay histogram showing HeLa cells stained with ab190200 (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 (ab190200, 1/500 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor^® 488 used at the same concentration and conditions as the primary antibody. 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.

This antibody gave a positive signal in HeLa fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

ab32537 staining ERK1 in wild-type HAP1 cells (top panel) and ERK1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32537 at 1/400 dilution and ab7291 at 1ug/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150117) at 2ug/ml (shown in pseudo-color red). Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal under the same testing conditions in HAP1 cells fixed with 4% formaldehyde (10 min).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Intracellular Flow Cytometry analysis of Jurkat cells labelling ERK1 with purified ab32537 at a dilution of 1/30 (red). Cells were fixed with 4% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

IHC image of ab32537 staining ERK1 in Human tonsil formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32537, 2μg/ml dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervix carcinoma tissue labelling ERK1 with purified ab32537 at a dilution of 1/50. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue labelling ERK1 with unpurified ab32537.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

Blocking buffer and concentration :  5% NFDM/TBST.  Diluting buffer and concentration :  5% NFDM/TBST. This blot was developed using a high sensitivity ECL substrate. ab181602 was used as a loading control. This data was developed using the same antibody clone in a different buffer formulation (ab32537).

All lanes:

Western blot - Anti-ERK1 antibody [Y72] (<a href='/products/primary-antibodies/erk1-antibody-y72-ab32537'>ab32537</a>) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Mouse heart lysate at 20 µg

Lane 3:

Mouse kidney lysate at 20 µg

Lane 4:

Mouse spleen lysate at 20 µg

Lane 5:

Raw 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 6:

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

Secondary

All lanes:

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

Predicted band size: 43 kDa

Observed band size: 43 kDa

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Exposure time: 180s

• IP

Unknown

Immunoprecipitation - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

ab32537 (purified) at a dilution of 1/20 immunoprecipitating ERK1 in Jurkat whole cell lysate.

Lane 1 (input) : Jurkat whole cell lysate (10μg)

Lane 2 (+) : ab32537 + Jurkat whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32537 in Jurkat whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32537).

All lanes:

Immunoprecipitation - Anti-ERK1 antibody [Y72] (<a href='/products/primary-antibodies/erk1-antibody-y72-ab32537'>ab32537</a>)

Predicted band size: 43 kDa

Observed band size: 44 kDa

false

• WB

Lab

Western blot - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

This data was developed using the same antibody clone in a different buffer formulation (ab32537).

Lanes 1- 2 : Merged signal (red and green). Green - ab32537 observed at 43 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32537 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. ab32537 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 [Y72] (<a href='/products/primary-antibodies/erk1-antibody-y72-ab32537'>ab32537</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='/products/cell-lines/human-mapk3-erk1-knockout-hek-293t-cell-line-ab266519'>ab266519</a>)

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

• WB

CiteAb

Western blot - Anti-ERK1 antibody [Y72] - BSA and Azide free (AB214168)

ERK1 western blot using anti-ERK1 antibody [Y72] - BSA and Azide free ab214168. Publication image and figure legend from Clift, D., McEwan, W. A., et al., 2017, Cell, PubMed 29153837.

ab214168 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab214168 please see the product overview.

Time Frame for Protein Depletion by Trim-Away, Related to Figure 5(A–F) NIH 3T3-mCherry-TRIM21 cells were taken up into the Neon Pipette Tip and either electroporated or not (mock). Percentage of dead cells was determined using the trypan blue exclusion assay (A). For long-term analysis of cellular behavior cells were imaged every 15 min for 70 h following electroporation (B-F). For the growth curves cell density was normalized to the initial density once cells had adhered (D). For adherence analysis the percentage of spread cells was quantified in each frame (B and C). Doubling times (E and F) were calculated taking the 5 h time point as reference, because cells were fully adhered then. Data from three independent experiments. Error bars show s.d. P values were calculated with Student’s t test. See also Movie S4.(G and H) HEK293T-mCherry-TRIM21 cells were electroporated with either control IgG, anti-ERK1 or anti-IKKα antibodies and whole cell lysates harvested at the indicated times after electroporation for immunoblot.(I–M) NIH 3T3-mCherry-TRIM21 cells were electroporated with control IgG or anti-Pericentrin antibody (BD611815) and 3 hours later analyzed for Pericentrin (I and K) or Cdk5rap2 (J and L) localization or cell lysates were immunoblotted for the indicated proteins (M). Merge also shows DNA stained with Hoechst. Number of cells in brackets. Scale bars, 5 μm. Data from two independent experiments. P values were calculated with Fisher’s exact (K) or Student’s t test (L).

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