Briefly, total protein was extracted and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes. The protein bands were probed with antibodies against AHNAK (Santa Cruz Biotechnology), Akt, phospho-Akt (Ser473), ERK1/2, phospho-ERK1/2 (Tyr202/Y204), phospho-c-Raf (Ser296), phospho-MEK1/2 (Ser221) (Cell Signaling Technology, Beverly, MA), c-myc, Wnt-1 and β-actin (Abcam, Cambridge, UK) overnight at 4 °C followed by incubation with HRP-conjugated second antibodies (Santa Cruz, CA, USA) (1:3500) and detected by enhanced chemiluminescence. The dilutions used for the anti-AHNAK and anti-β-actin antibodies were 1:200 and 1:5000, respectively. The dilution used for the other antibodies was 1:1000. β-actin was used as the protein-loading control.
Phospho mek1 2 ser221
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, China, United States
Phospho-MEK1/2 (Ser221) is a primary antibody that specifically recognizes MEK1/2 phosphorylated at serine 221. This antibody can be used to detect the activation state of the MEK1/2 protein.
Automatically generated - may contain errors
Lab products found in correlation
Erk1 2, by Cell Signaling Technology (1 mentions)
Hrp conjugated second antibody, by Santa Cruz Biotechnology (1 mentions)
β actin, by Abcam (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Ahnak, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Mek1 2, by ABclonal (1 mentions)
Smurf1, by Bioss Antibodies (1 mentions)
Erk1 2, by Proteintech (1 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Gapdh, by Proteintech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence western blot substrate, by Thermo Fisher Scientific (1 mentions)
3 protocols using phospho mek1 2 ser221
1
Western Blot Analysis of Cellular Signaling
2
Western Blot Profiling of Intracellular Signaling
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Cells were lysed with protein lysis buffer (Solarbio, R0020) containing cocktail and then separated by SDS-PAGE gels, and transferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After being blocked with 5% nonfat milk, the membrane was incubated with a specific primary antibody at room temperature for 1 h, and then washed and incubated with a secondary antibody (Proteintech, USA) for 1 h. Western blotting was performed by anti-MEKK2 (Proteintech, 55106-1-AP), MEK1/2 (ABclonal, A4868), SMURF1 (Bioss, bs-9391R), STK38 (Huabio, ER60144), ERK1/2 (Proteintech, 11257-1-AP), UB (Cell Signaling Technology, #3936), Phospho-MEK1/2 (Ser221) (Cell Signaling Technology, #2338), Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling Technology, #8544), CAPG (Bioss, bsm-60,451 M), CDK4 (AtaGenix Laboratories Co., Ltd.(Wuhan), Wuhan, PR China, PA9437H), Cyclin D1 (Yeasen, 30026ES50), BCL2 (Shanghai hengyuan biological technology co., LTD, A-01203), CAPG (Proteintech, 10194-1-AP), FLAG (SMART, /smart-lifesciences, SLAB0101), HA (Yeasen, 30702ES20), HIS (Yeasen, 30401ES10). β-ACTIN, GAPDH, or TUBULIN (Proteintech, USA) were used as loading controls. All antibodies were diluted as required by the manufacturer (New Cell & Molecular Biotech, WB100D). The antibodies used in this study are listed in Table S2 .
3
Profiling ERK1/2 and MEK1/2 Phosphorylation
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Phosphorylation of ERK1/2 and MEK1/2 were further validated by Western blot analysis. Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cat. No. 4320, 1: 1000) and Phospho-MEK1/2 (Ser221) (Car: 2338, 1: 1000) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The glyceraldehyde-3-phosphate dehydrogenase (ab8245, 1: 5000) antibody was from Abcam, and the HRP-conjugated secondary antibody (Cat. No. SA00001-2, 1: 5000) was from Proteintech. Protein lysates were prepared using RIPA lysis buffer and the concentration was determined using the BCA kit. Then, 25 μg total protein was collected and separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (EMD Millipore Corp., Billerica, MA, USA). Next, the membranes were blocked using skimmed milk at room temperature for 30 min, washed, and incubated with the primary antibodies at 4°C overnight, and then with the secondary antibody at room temperature for 1 h. The blots were developed using the enhanced chemiluminescence Western blot substrate (Thermo Fisher). Relative protein expression was determined using Image J software.
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