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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [1]
- Immunocytochemistry [1]
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- Product number
- 711612 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-MEK1 (Thr386) Recombinant Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with Monkey, Bovine and Pig.
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1) and HEK-293 treated with EGF (100 ng/mL for 30 mins) (Lane 2). The blots were probed with Anti-MEK1 (pT386) Recombinant Rabbit Polyclonal Antibody (Product # 711612, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 43 kDa band corresponding to MEK1 (pT386) was observed across the cell lines tested. 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® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). 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
- For immunofluorescence analysis, HeLa cells were fixed and permeabilized for detection of endogenous Mek1 pT386 using Anti- Mek1 pT386 Recombinant Rabbit Polyclonal Antibody (Product # 711612, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of Mek1 pT386 (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating localization of Mek1 pT386 in dividing cells. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
