- 44-460G - Invitrogen Antibodies MAP2K1 antibody

Chow HY, Dong B, Valencia CA, Zeng CT, Koch JN, Prudnikova TY, Chernoff J
Nature communications 2018 Aug 27;9(1):3473

Chow HY, Dong B, Duron SG, Campbell DA, Ong CC, Hoeflich KP, Chang LS, Welling DB, Yang ZJ, Chernoff J
Oncotarget 2015 Feb 10;6(4):1981-94

Mao WF, Shao MH, Gao PT, Ma J, Li HJ, Li GL, Han BH, Yuan CG
Acta pharmacologica Sinica 2012 Oct;33(10):1311-8

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Peptide Competition and Phosphatase Treatment Extracts of NIH3T3 cells untreated (1) or treated with 50 ng/mL PDGF for 15 minutes (2-7) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was either left untreated (1-5, 7) or treated with lambda phosphatase (6), blocked with a 4% BSA-TBST buffer for one hour at room temperature, then incubated with the MEK1 [pS298] antibody (Product # 44-460G) for two hours at room temperature in a 1% BSA-TBST, following prior incubation with: no peptide (1, 2, 6, 7), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphoserine-containing peptide (4) or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to MEK1 [pS298] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MEK1 (pS298) was performed by loading 20 µg of HeLa (lane1), U-87 MG (lane2), PC-3 (lane3) and A431 (lane4) cell lysate using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® 2 Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. MEK1 (pS298) was detected at ~ 43 kDa using MEK1 (pS298) Rabbit Polyclonal Antibody (Product # 44-244G) at 1:1000 dilution in 5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: MEK1 (pS298) phosphospecific antibody. A549 cells plated on fibronectin, serum starved overnight then stimulated with serum for 1 hour. Green = MEK1 (pS298), red = actin, blue = Hoechst 33342.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Phospho-MEK1 pSer298 Antibody was done on 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-MEK1 pSer298Antibody (Product # 44-460G) at 1:250 dilution in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 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 Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic and nuclear localization. Panel e is a no primary antibody control. The images were captured at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of MEK1 (pS298) showing staining in the cytoplasm of paraffin-embedded human colon carcinoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a MEK1 (pS298) polyclonal antibody (Product # 44-460G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of MEK1 (pS298) showing staining in the cytoplasm of paraffin-embedded human kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a MEK1 (pS298) polyclonal antibody (Product # 44-460G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of MEK1 [pS298] was done on A549 cells treated with EGF (200ng/mL, 10 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with MEK1 [pS298] Rabbit Polyclonal Antibody (44460G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 The effect of Pak inhibitors on Erk and Akt-S6 signaling pathways KT21 or Ben-Men cells were treated with inhibitors for 72 hours as described in Figure 2 . Following SDS/PAGE and transfer to PVDF membranes, expression levels of Pak, Mek, Erk, Akt, S6, and beta-Catenin were assessed by immunoblot using total and phospho-specific antibodies. GADPH was used as loading control.

44-460G