The total proteins of cells were extracted using the RIPA lysis buffer (Beyotime, Shanghai, China) with Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). Protein samples were separated in sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinylidene fluoride (PVDF) filter membranes (Millipore, USA) for immune-hybridization. After 1 h of blocking in PBST (phosphate-buffered saline containing 0.05% Tween-20 and 5% non-fat milk powder), the membranes were incubated with one of the following primary antibodies with corresponding concentration: RNF8, 1:500, (Santa Cruz Biotech, CA, USA), EMT kit (1:2000, Cell Signaling Technologies (CST), Danvers, MA, USA), beta-actin (Santa Cruz Biotech,1:4000), Secondary antibodies were Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:4000, ZB-2305, ZSGB-Bio, Beijing, China) or anti-Rabbit IgG (1:4000, ZB-2301, ZSGB-Bio). Subsequently, band visualization was performed using electro-chemiluminescence (ECL) and detected by the Digit imaging system (Thermo, Japan).
Emt kit
Manufactured by Cell Signaling Technology
Sourced in United States
The EMT kit is a laboratory equipment designed to facilitate the isolation and enrichment of epithelial-mesenchymal transition (EMT) cells from biological samples. It provides the necessary tools and reagents to perform this specialized cell separation procedure.
Automatically generated - may contain errors
Lab products found in correlation
Digit imaging system, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Pvdf filter membrane, by Merck Group (3 mentions)
Protease inhibitor, by Roche (2 mentions)
Phosphatase inhibitor, by Roche (2 mentions)
Hrp conjugated anti mouse igg, by ZSGB-BIO (2 mentions)
Anti rabbit igg fc, by ZSGB-BIO (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Cleaved casepase3, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Akr1c1, by Abcam (1 mentions)
Anti mmp 9, by Immunoway (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Anti tgf β1, by Cell Signaling Technology (1 mentions)
Mouse anti β tubulin, by Cell Signaling Technology (1 mentions)
5 protocols using emt kit
1
Protein Expression Analysis by Western Blot
2
Quantitative Western Blot Analysis of EMT Markers
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Total proteins were extracted with RIPA lysis buffer (Beyotime) with Protease Inhibitor Cocktail (Roche) and Phosphatase Inhibitor Cocktail (Roche). Protein samples were separated in sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinylidene fluoride (PVDF) filter membranes (Millipore, USA). After blocking in phosphate buffered saline (PBS) containing 0.05% Tween-20 and 5% non-fat milk powder, the membranes were incubated with the following primary antibodies: RNF8 (Santa Cruz Biotech, 1:500), EMT kit (Cell Signaling Technology, 1:2000), beta-actin (Santa Cruz Biotech, 1:4000), Secondary antibodies were Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (ZB-2305, ZSGB-Bio, 1:4000) or anti-Rabbit IgG(Fc) (ZB-2301, ZSGB-Bio, 1:4000). Subsequent visualization was detected by Digit imaging system (Thermo, Japan), the gray level of the bands was quantitated by ImageJ software.
3
Immunoblotting and Immunohistochemistry Protocols
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Antibodies used in WB and IHC experiments were following: MGLL (Abcam, ab234701), CDK4 (Cell Signaling Technologies, #12790), Cyclin D1 (Cell Signaling Technologies, #2978), cleaved-PARP (Cell Signaling Technologies, #5625), Bcl2 (Cell Signaling Technologies, #4223), cleaved-casepase3 (Cell Signaling Technologies, #9664), EMT kit(Cell Signaling Technologies, #9782), β-actin (Cell Signaling Technologies, #4970), Ki67 (Abcam, ab92742),AKR1C1(Abcam,ab179448), mouse IgG (Cell Signaling Technologies, #7076), and rabbit IgG (Cell Signaling Technologies, #7074). Medroxyprogesterone acetate(MPA) and ABX-1431 were obtained from Abcam and Selleck, respectively, and were both diluted in DMSO.
4
Western Blot Analysis of Cellular Proteins
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The cultured cells were rinsed with cold PBS before treated with cell lysis buffer aids (2 × loading buffer, 2 µg/ml aprotinin, 1 mM PMSF, 2 mM β-mercaptoethanol) at 100 °C for 10 min. Then the mixture was centrifuged under 4 °C at 12000 rpm for 10 min. About 25–30 μg of protein was loaded in each lane, separated by 10% SDS-PAGE, and transferred to the PVDF membrane. The membrane was blocked with 5% nonfat milk powder for 1 h at room temperature before overnight incubation with primary antibodies at 4 °C, followed by the secondary antibody. The blots were incubated with primary antibodies rabbit anti-MeCP2 (Cell Signaling, CAT 3456), rabbit anti-Furin (Proteintech, CAT 18413-1-AP), anti-TGF-β1 (Cell Signaling, CAT 3709), anti-TGF-βR (Cell Signaling, CAT 3712), anti-MMP2 (Immunoway, CAT YT2798), anti-MMP9 (Immunoway, CAT YT1892), EMT Kit (Cell Signaling, CAT 9782), Smad2/3 Antibody Sampler Kit (Cell Signaling, CAT 12747), mouse anti-Flag (SIGMA, CAT 6631), mouse anti-β-Tubulin (Cell Signaling, CAT4466).
5
Protein Expression Analysis via SDS-PAGE
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Total proteins of cells were extracted using RIPA lysis buffer (Beyotime) with Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). Protein samples were separated in sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinylidene fluoride (PVDF) filter membranes (Millipore, USA) for immune-hybridization. After 1 hour blocking in PBST (phosphate buffered saline containing 0.05% Tween-20 and 5% non-fat milk powder), the membranes were incubated with one of the following primary antibodies with corresponding concentration: RNF8 (Santa Cruz Biotech, 1:500), EMT kit (Cell Signaling Technology, 1:2000), beta-actin (Santa Cruz Biotech,1:4000), Secondary antibodies were Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (ZB-2305,ZSGB-Bio,1:4000) or anti-Rabbit IgG (ZB-2301, ZSGB-Bio, 1:4000). Subsequently, band visualization was performed by electro-chemiluminescence (ECL) and detected by Digit imaging system (Thermo, Japan).
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