Preprocessed samples were added to the RIPA lysate solution (P0013B; Beyotime, China), and the supernatants were collected after centrifugation. Samples were loaded onto sodium dodecyl sulfate-polyacrylamide gels (3250GR500; neoFROXX, Einhausen, Germany). Proteins were transferred to polyvinylidene difluoride membranes (WGPVDF22; Servicebio, China) after electrophoresis in transfer buffer for 5 min. The membranes were then incubated with primary antibodies (1:5000 dilution) targeting CD31 (AF6191, Affinity, USA), Vimentin (BF8006, Affinity), α-SMA (AF1032, Affinity), slug (350,136, ZENBIO, China), snail (AF6032, Affinity), twist (AF4009, Affinity), LC3II (3868S, CST, USA), p62 (AF5384, Affinity), beclin 1 (AF5128, Affinity), AMPK (AF6423, Affinity), p-AMPK (AF3423, Affinity), mTOR (AF6308, Affinity), and p-mTOR (AF3308, Affinity). Horseradish peroxidase-conjugated secondary antibodies at a 1:10,000 dilution were added after washing. The membranes were then viewed with an automatic darkroom exposure instrument (JS-M6P; P&Q, China) and varying luminous intensities were used for optimal exposure.
Af4009
Manufactured by Affinity Biosciences
Sourced in United States
The AF4009 is a compact and versatile laboratory instrument designed for sample preparation and handling. It features a streamlined and user-friendly interface, enabling efficient processing of a wide range of sample types. The core function of the AF4009 is to facilitate straightforward sample processing and preparation tasks within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Af1032, by Affinity Biosciences (1 mentions)
Ripa lysate solution, by Beyotime (1 mentions)
Af6191, by Affinity Biosciences (1 mentions)
Bf8006, by Affinity Biosciences (1 mentions)
Af6032, by Affinity Biosciences (1 mentions)
Af6308, by Affinity Biosciences (1 mentions)
Af6423, by Affinity Biosciences (1 mentions)
Af3423, by Affinity Biosciences (1 mentions)
Af3308, by Affinity Biosciences (1 mentions)
Wgpvdf22, by Wuhan Servicebio Technology (1 mentions)
Af5128, by Affinity Biosciences (1 mentions)
Af5384, by Affinity Biosciences (1 mentions)
Pb9439, by Boster Bio (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Gb23303, by Wuhan Servicebio Technology (1 mentions)
Gb23301, by Wuhan Servicebio Technology (1 mentions)
Pierce ecl western blotting substrate kit, by Thermo Fisher Scientific (1 mentions)
Af0131, by Affinity Biosciences (1 mentions)
Af7021, by Affinity Biosciences (1 mentions)
Af4039, by Affinity Biosciences (1 mentions)
5 protocols using af4009
1
Western Blot Analysis of Cell Markers
2
Western Blot Analysis of Protein Expression
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Protein was extracted from the cells were resolved by SDS-PAGE and then transferred to PVDF membranes (IPVH00010, Millipore), and then incubated with primary antibodies diluted in blocking buffer at 4 °C overnight. The following primary antibodies were used: Mouse Anti-β-Actin (HC201, TransGen Biotech, 1/2000), HRP conjugated Goat Anti-Mouse IgG (H + L) (GB23301, Servicebio, 1/2000), Rabbit Anti EMP3 (DF14661, Affinity, 1/1000), Rabbit Anti TWIST1(AF4009, Affinity, 1/1000), Rabbit Anti SNAI2 (PB9439, Boster, 1/1000), Rabbit Anti FOS (AF5354, Affinity, 1/1000), Mouse Anti-VIM (60330–1-Ig, Proteintech,1/10000), HRP conjugated Goat Anti-Rabbit IgG (H + L) (GB23303, Servicebio, 1/2000), HRP conjugated Goat Anti-Mouse IgG (H + L) (GB23301, Servicebio, 1/2000)was used. Finally, the antigen–antibody reaction was visualized by the enhanced Pierce ECL Western blotting substrate kit (Thermo Scientific/ Pierce, Rockford, IL, USA).
3
Protein Expression Analysis via Western Blot
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The BCA kit (pc0020, Solarbio, China) was utilized to quantify the protein isolated from cells, followed by being separated with the 12% SDS-PAGE. The separated protein was transferred from the gel to the PVDF membrane, which was further introduced with 5% skim milk. Then, the membrane was introduced with the primary antibody against PI3K (1:1000, AF6241, Affinity, USA), p-AKT (1:1000, AF0016, Affinity, USA), AKT (1:1000, AF6261, Affinity, USA), OPN (1:2000, AF0227, Affinity, USA), Twist (1:1000, AF4009, Affinity, USA), E-cadherin (1:2000, AF0131, Affinity, USA), N-cadherin (1:1000, AF4039, Affinity, USA), and GAPDH (1:10000, AF7021, Affinity, USA). The second antibody (1:6000, 7074, CST, USA) was subsequently added to be incubated for 90 min. Finally, ECL reagent was added to expose the bands, which were further quantified with the Image J software.
4
Western Blot Analysis of EMT Markers
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Cells were lysed in a lysis buffer after washing twice with ice-cold PBS, followed by quantification of the total protein concentrations using a BCA kit (Beyotime Biotechnology, Shanghai, China). Next, 20µg of total protein was boiled for 5 min and resolved using 10% polyacrylamide gel electrophoresis. Proteins were then transferred to a polyvinylidene fuoride (PVDF) membrane. The membranes were blocked in no fat milk and then incubated with primary antibodies overnight at 4 °C. The primary antibodies included anti-ANK3 (1:1000, A20299, ABclonal, Wuhan, China), anti-E-cadherin (1:1000, ab231303, Abcam, Cambridge, MA, USA), anti-vimentin (1:1000, AF7013, Affinity Biosciences, Jiangsu, China), anti-snail (1:500, ab180714, Abcam, Cambridge, MA, USA), anti-twist (1:1000, AF4009, Affinity Biosciences, Jiangsu, China), and anti-GAPDH (1:1000, AB-P-R 001, Xianzhi Biotech, Hanzhou, China) at 37°C overnight. On the next day, membranes were incubated with the secondary antibodies: anti-Mouse IgG-HRP (1:50000, Boster, Wuhan, China) and anti-Rabbit IgG-HRP (1:50000, Boster, Wuhan, China). Finally, the specific protein bands on the membranes were detected using an enhanced chemiluminescence kit (Beyotime, Shanghai, China).
5
Immunofluorescence Imaging of TWIST1 in Tumor Sections
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The tumor masses were embedded in paraffin and sliced into 10 μm thick sections. The studies involving human samples were approved by Shandong Provincial ENT Hospital Ethical Committee (20220404). The sections were then blocked with PBT-1 for 60 minutes at room temperature, and incubated overnight at 4°C with primary antibody (anti-TWIST1, 1:100, AF4009; Affinity, Jiangsu, China). The following morning, the sections were incubated with an associated secondary antibody conjugated with Alexa Fluor 647 (1:1000; Invitrogen, Carlsbad, CA) and DAPI (1:1000, D9542; Sigma-Aldrich, St. Louis, MO) for 1 hour in the dark at room temperature. The fluorescently labeled sections were observed using a laser scanning confocal microscope (Leica SP8; Leica, Wetzlar, Germany).
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