The total protein from cultured cells and tumors were obtained with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA), separated by 10% polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). The protein membrane was incubated overnight at 4°C with primary antibodies (anti-pERK1/2, anti-total ERK1/2, anti-pMEK1/2, anti-MEK1/2, anti-pMER1/2, anti-pSTAT3, anti-STAT3, anti-pAKT, anti-AKT, and anti-GAPDH antibody, Abcam, Cambridge, MA, USA). We used the enhanced chemiluminescence (Thermo Scientific, Waltham, MA) to detect protein bands and semi-quantified the band density with an ImageJ analysis software (National Institutes of Health).
Anti mek1 2
Manufactured by Abcam
Sourced in United States
Anti-MEK1/2 is a primary antibody that binds to the MEK1 and MEK2 proteins. MEK1 and MEK2 are kinases that play a central role in the MAPK/ERK signaling pathway. This antibody can be used for applications such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti p erk1 2, by Abcam (4 mentions)
Anti pmek1 2, by Abcam (4 mentions)
Anti erk1 2, by Abcam (3 mentions)
Anti phospho mek1 2, by Abcam (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Anti gapdh antibody, by Abcam (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Anti akt, by Abcam (2 mentions)
Anti total erk1 2, by Abcam (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Anti p akt, by Abcam (1 mentions)
Anti p stat3, by Abcam (1 mentions)
Anti stat3, by Abcam (1 mentions)
Anti ppka, by Abcam (1 mentions)
Anti pka, by Abcam (1 mentions)
Anti psapk jnk, by Abcam (1 mentions)
Anti bcl xl, by Abcam (1 mentions)
Anti p p38 mapk thr180 tyr182, by Cell Signaling Technology (1 mentions)
Electrophoresis reagents, by Bio-Rad (1 mentions)
9 protocols using anti mek1 2
1
Western Blot Analysis of Signaling Proteins
2
Quantitative Immunoblotting for Signaling Proteins
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The following reagents and materials were used: anti-ALDH1B1 (ref. sc-374090) was purchased from Santa Cruz; anti-PI16 (ref. PAS 31882) from Thermo; anti-P70 S6K beta (ref: ab184551) from Abcam; and anti-Bcl-xL (ref. 2764), anti-pAkt (Ser473) (ref. 4060), anti-Akt (ref. 4685), anti-pMEK1/2 (Ser217/221) (ref. 9154), anti-MEK1/2 (ref. 9126), anti-pERK1/2 (Thr202/Tyr204) (ref. 4370), anti-ERK1/2 (ref. 9102), anti-pPKA (Thr197) (ref. 5661), anti-PKA (ref. 4782), anti-pSEK1/MKK4 (Ser257/Thr261) (ref. 9156), anti-SEK1/MKK4 (ref. 9152), anti pSAPK/JNK (Thr183/Tyr185) (ref. 9255), anti pSAPK/JNK (ref. 9252S), anti pMKK3-6 (Thr183/Tyr185) (ref. 9231), anti MKK3 (ref. 5674), anti p-p38 MAPK (Thr180/Tyr182) (ref. 4511), anti p38 MAPK (ref. 9212) were purchased from Cell Signaling. Electrophoresis reagents were purchased from Biorad and trypsin from Promega.
3
Western Blot Analysis of MAPK and AKT Signaling
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LUAD cells were lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Cell lysates were electrophoresed in 12% SDS-PAGE and then transferred onto a nitrocellulose membrane (MerrckMillipore, Ireland). Membranes were blocked with TBST (Tris-buffered saline, 0.1% Tween-20) containing 5% skim milk for 2 h and were incubated with the indicated antibodies, including anti-MEK1/2 (Abcam, CAT# ab178876);anti-Phospho-MEK1/2 (CST, CAT# 9154 T);anti-ERK1/2 (CST, CAT#4695 T); anti-Phospho-ERK1/2(CST, CAT#4370 T); anti-AKT (CST, CAT#4691 T); anti-Phospho-AKT (CST, CAT#4060 T);anti-STAT3 (Huaan Biotech, ET1607-38); anti-Phospho-STAT3 (CST, CAT#9145 T) overnight at 4 °C. Then, the membranes were washed with TBST and incubated with a peroxidase-conjugated second antibody for 1 h. The protein bands were detected by enhanced chemiluminescence.
4
Western Blot Analysis of Mitochondrial Proteins
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Protein was extracted using SDS lysis buffer (Beyotime Biotechnology, Shanghai, China), and equal amounts of total protein were separated by 10% SDS–PAGE and then transferred to nitrocellulose membranes (Millipore, USA). The membrane was blocked with 5% nonfat powdered milk (Sangon, China) in TBST at room temperature for one hour, incubated with primary antibodies overnight at 4 °C, and then washed with TBST followed by a 1 h incubation with secondary antibodies. The protein bands were visualized by an enhanced chemiluminescence detection kit (Tanon, China). Semiquantitative analysis of the protein density by western blotting was performed using ImageJ (Version 1.5.3). The following antibodies were used at a 1:1000 dilution: anti-SLC25A5 (Cat. #14671, Cell Signaling), anti-SLC25A24 (Cat. #221120, Abcam), anti-COX IV (Cat. #202554, Abcam), anti-phospho-MEK1/2 (Cat. #178876, Abcam), anti-MEK1/2 (Cat. #278564, Abcam), anti-phospho-ERK1/2 (Cat. #201015, Abcam), anti-ERK1/2 (Cat. #184699, Abcam), anti-caspase-1 (Cat. #207802, Abcam), anti-IL-1β (Cat. #254360, Abcam), anti-IL-18 (Cat. #243091, Abcam), anti-gasdermin D (Cat. #219800, Abcam), anti-β-actin (Cat. #4970, Abcam) and anti-GAPDH (Cat. #2118, Cell Signaling).
5
Western Blotting of Mitochondrial Proteins
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The mitochondrial pellet was extracted from the cell culture using the EXkineTM Mitochondrion Extraction Kit (Abbkine USA) and resuspended in RIPA lysis buffer, which was frozen for Western blotting. Total protein using RIPA lysis buffer (Beyotime Institute of Biotechnology, China) containing 1% protease inhibitor. A 5 × loading buffer was added, and the mixture was boiled at 100 °C for 10 min. Proteins were transferred to polyvinylidene fluoride membranes (Millipore Corporation, USA). The membranes were blocked with 5% bovine serum albumin (BSA), followed by incubation with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-phosphorylated (p)-MEK1/2 (1:10,000; Abcam, UK), anti-MEK1/2 (1:10,000; Abcam), anti-phosphorylated (p)-ERK1/2 (1:10,000; Abcam), anti-ERK1/2 (1:10,000; Abcam), anti-cleaved caspase-3 (1:1000; Cell Signaling, USA), anti-Bcl-2 (1:2000; Abcam), anti-Bax (1:2000; Abcam), and anti-GAPDH (1:5000; Abcam). Membranes were then incubated with HRP-conjugated secondary antibodies (1:5000; Abcam) for 1 h at room temperature. ECL reagent (Beyotime Institute of Biotechnology) was used for immunodetection and visualization using a Bio-Image Analysis System (Syngene, USA). Densitometric quantification was performed using ImageJ after normalization with GAPDH.
6
Western Blot Analysis of Protein Expression
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Total protein was extracted from tissues or cells. Protein lysates (50 μg/lane) were separated by 10% sodium lauryl sulfate-polyacrylamide gels (SDS-PAGE) and transferred onto a nitrocellulose membrane (Sigma-Aldrich; Merck KGaA). The membranes were blocked in 5% nonfat milk for 1 hour at room temperature and incubated at 4 °C overnight with a primary antibody: anti-LUM, anti-ERK, anti-p-ERK, anti-MEK1/2, anti-p-MEK1/2 and anti-MMP-2 (Abcam, Cambridge, MA), anti-Cyclin D1 (BOSTER, Wuhan, China). β-actin (Abcam, Cambridge, MA) was used as an internal control. After incubation with the secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 hour at room temperature, the signals were visualized using the LI-COR Image Studio lite imaging system.
7
Western Blotting Protocol with Antibodies
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The protocol for Western blotting is described in detail in [18 (link)]. The following antibodies were used: anti-ERK1,2 (Santa Cruz Biotechnology, Heidelberg, Germany; sc-94), anti-phospho-ERK1,2 (Cell Signaling Technology (CST), Frankfurt am Main, Germany; no. 9101), anti-MEK1,2 (CST; no. 9122), anti-phospho-MEK1,2 (CST; no. 9154), anti-HA-tag (Abcam, Cambridge, UK; ab9110), anti-tet-Repressor (Merck, Darmstadt, Germany; AB3541), anti-HSP90β (Enzo Life Sciences, Lörrach, Germany; ADI-SPA-843), anti-α-tubulin (Bio-Rad, München, Germany; MCA78G). Secondary antibody F(ab')2 fragments coupled to horseradish peroxidase and specific for rabbit-IgG (no. 111-036-045), mouse-IgG (no. 115-036-072) or rat (no. 112-036-062) were obtained from Jackson ImmunoResearch, Newmarket, UK.
A freshly made solution of luminol (2.5 mM), p-coumaric acid (0.2 mM) and H2O2 (0.01%) in 100 mM Tris-HCl (pH 8.8) was used as reagent for chemiluminescent detection [19 (link)].
A freshly made solution of luminol (2.5 mM), p-coumaric acid (0.2 mM) and H2O2 (0.01%) in 100 mM Tris-HCl (pH 8.8) was used as reagent for chemiluminescent detection [19 (link)].
8
Signaling Pathways in KRAS-Driven Cancers
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MDA-MB-231 (KRASG13D), SW1116 (KRASG12A), SW480 (KRASG12V), Hela (KRASwt), SW48 (KRASwt) and Colo320 (KRASwt) cells were grown in Dulbecco’s modified Eagle medium with 10% foetal calf serum (FCS; all from Gibco/Invitrogen, USA). Trametinib and Deltarasin were from Selleckchem, Simvastatin was from Sigma, Leptomycin B was from Beyotime, China. MG132 and Doxycycline were from MedChemExpress. The following antibodies were used for WB, Co-IP, IF and IHC staining: Anti-YAP, anti-MEK1/2, anti-pERK1/2, anti-Lamin B, anti-IQGAP1 and anti-Ki-67 were all from Abcam. Anti-GAPDH and anti-HA were from Santa Cruz. Anti-LATS1 and anti-β-TrCP (WB/Co-IP) were from Cell Signaling Technology and anti-β-TrCP (IF) was from Sangon Biotech, China. Anti-pMEK1, anti-CYR61 and anti-Flag were from Sangon Biotech. Anti-KRAS was from Proteintech.
9
Immunoblot Analysis of Cell Signaling
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The total protein from cultured cells and tumors were obtained with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and separated by 10% polyacrylamide gel electrophoresis, followed by transferring onto polyvinylidene uoride membranes (Millipore, MA, USA). The protein membrane was incubated with primary antibodies (Anti-pERK1/2, anti-total ERK1/2, Anti-pMEK1/2, Anti-MEK1/2, and Anti-GAPDH antibody, Abcam, Cambridge, MA, USA) overnight at 4 °C. We used the enhanced chemiluminescence (Thermo Scienti c, Waltham, MA) to detect protein bands and semi-quanti ed the band density with an ImageJ analysis software (National Institutes of Health).
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