PA5-13036

antibody from Invitrogen Antibodies

Targeting: MAPK3 ERK1, p44erk1, p44mapk, PRKM3

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-13036

Provider

Invitrogen Antibodies

Product name

Phospho-ERK1 (Thr202, Tyr205) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with canine, mouse and rat based on sequence homology.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

400 μL

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of Phospho-ERK1 (Thr202, Tyr205) in extracts from Hela and NIH/3T3 cells. Samples were incubated with Phospho-ERK1 (Thr202, Tyr205) polyclonal antibody (Product # PA5-13036). Phospho-ERK1-T202/Y204 Antibody (upper) or Tubulin (lower).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg) of A-431 (1), A-431 treated with EGF (200 ng/mL for 10 minutes) (2), A-431 treated with Afatinib followed by EGF (0.5 uM of Afatinib for 6hrs, 200 ng/mL for 10 minutes) (3), A549 (4) and A549 treated with EGF (200 ng/mL for 10 minutes) (5). The blot was probed with Anti-Phospho-ERK1/ERK2 (Thr202, Tyr205) Rabbit Polyclonal Antibody (Product# PA5-13036, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). 44, 42 kDa band corresponding to Phospho-ERK1/ERK2 (Thr202, Tyr205) was detected EGF treatment across cell lines tested and pre-treatment with Afatinib (antagonist) resulted in inhibition of Phospho-ERK1/ERK2 (Thr202, Tyr205) in A-431 cell line upon EGF treatment. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by wet transfer method. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed by loading 30 µg of A-431 (Lane 1), A-431 treated with EGF (200 ng/mL for 10 minutes) (Lane 2), A-431 treated with Afatinib followed by EGF (0.5 uM of Afatinib for 6hrs, 200 ng/mL for 10 minutes) (Lane 3), A-431 EGFR KO (Lane 4) A-431 EGFR KO treated with EGF (200 ng/mL for 10 minutes) (Lane 5), A-431 EGFR KO treated with Afatinib followed by EGF (0.5 uM of Afatinib for 6hrs, 200 ng/mL for 10 minutes) (Lane 6) whole cell extracts using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex Protein Standard (Product # LC5800). Proteins were transferred to a nitrocellulose membrane using iBlot® Dry Blotting System (IB21001) and blocked with 5% skim milk for 1 hour at room temperature. The blot was probed with Anti-Phospho-ERK1/ERK2 (Thr202, Tyr205) Rabbit Polyclonal Antibody (Product# PA5-13036, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). 44, 42 kDa band corresponding to Phospho-ERK1/ERK2 (Thr202, Tyr205) was detected in EGF treatment in control cell line (lane1 and 2) and not in EGFR knockout cell line (lane 4 and 5). Pre-treatment with Afatinib (antagonist) resulted in inhibition of Phospho-ERK1/ERK2 (Thr202, Tyr205) upon EGF treatment (lane 3 and 6).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Phospho-ERK1 (Thr202, Tyr205) was performed using 90% confluent log phase A-431 cells treated with 200 ng/mL of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-ERK1 (Thr202, Tyr205) Rabbit Polyclonal Antibody (Product # PA5-13036) at 5 µg in 0.1% BSA and incubated overnight at 4 degree Celsius and then labelled with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:100). Panel d represents the merged image showing membrane and cytoplasmic localization. Panel e represents cells treated with antagonist, Afatinib (1µM for 6hrs) followed by EGF (200 ng/mL for 10 minutes), showing no signal. Panel f shows untreated cells with nuclear and cytoplasmic staining. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Dot blot analysis of Phospho-ERK1 (Thr202, Tyr205). Samples were incubated with Phospho-ERK1 (Thr202, Tyr205) polyclonal antibody (Product # PA5-13036) using a dilution of 0.5 µg/mL. 50 ng of Bisphospho-peptide or Non Phosphorylated peptide per dot were adsorbed on nitrocellulose membrane.