After different proportions of MSC and MG63 were co-cultured to a predetermined time, the supernatant of the upper and lower chambers of the 6-well cell culture plate was aspirated, and cells were cleaned twice with PBS. A protein extraction kit (SD-001, Invent Biotechnologies, Inc, China) was used for protein extraction. Lysis buffer was added and spilled on the ice for 5 min. Then, the protein lysate was filtered through a centrifugal column and placed into a centrifuge for 10 minutes under the condition of 10000 rpm and 4°C, and the clear fluid in the collection tube is the protein. NanoDrop 2000 was used to measure protein concentration. After that, the protein was prepared with a 5x loading buffer in proportion, and the sample was performed using publishing protocols [16 (link), 17 (link)] with antibodies directed against α-smooth muscle actin (α-SMA, ab5694, Abcam, Cambridge, UK), E-cadherin (ab212059, Abcam), N-cadherin (AF4039, Affinity), Slug (ab51772, Abcam), Snail (ab216347, Abcam), and Vimentin (ab92547, Abcam); the dilution of these antibodies was 1 : 1000, and β-actin (ab8227, Abcam) and GAPDH (ab9485, Abcam) ratio of these two antibodies was 1 : 100000.
Af4039
Manufactured by Affinity Biosciences
Sourced in United Kingdom, United States
AF4039 is a high-precision laboratory instrument designed for the analysis of macromolecular and particulate samples. It utilizes the principles of Asymmetric Flow Field-Flow Fractionation (AF4) to separate and characterize a wide range of materials, including proteins, nanoparticles, and polymers.
Automatically generated - may contain errors
Lab products found in correlation
Af0131, by Affinity Biosciences (3 mentions)
Ab9485, by Abcam (2 mentions)
Af7022, by Affinity Biosciences (2 mentions)
Af7021, by Affinity Biosciences (2 mentions)
Ab212059, by Abcam (1 mentions)
Ab51772, by Abcam (1 mentions)
Ab216347, by Abcam (1 mentions)
Sd 001, by Invent Biotechnologies (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab5694, by Abcam (1 mentions)
Xylene, by Merck Group (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Af0198, by Affinity Biosciences (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
Cx31 lv320 microscope, by Olympus (1 mentions)
Af0227, by Affinity Biosciences (1 mentions)
6 protocols using af4039
1
Protein Expression Analysis of MSC-MG63 Co-culture
2
Immunohistochemical Analysis of Xenograft Tumors
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Immunohistochemistry (IHC) assay was designed to analyze the expression of nuclear proliferation marker (Ki‐67), Cleaved‐caspase 3, E‐cadherin and N‐cadherin in the transplanted tumors from xenograft tumor model assay as instructed.26 The tumors were cut into the 4‐µm‐thick sections, and embedded into paraffin, followed by the heating at 60°C. Twenty minutes later, the sections were deparaffinized and hydrated with xylene (Millipore) as well as ethanol (Millipore). After that, the sections were dipped in the sodium citrate (Millipore) and heated to conduct the antigen retrieval. After being immersed in Hydrogen Peroxide (Millipore) for 10 min, the sections were incubated with the primary antibodies against Ki‐67 (AF0198; 1:100; Affinity), Cleaved‐caspase 3 (AF7022; 1:100; Affinity), E‐cadherin (AF0131; 1:100; Affinity) and N‐cadherin (AF4039; 1:100; Affinity) as well as secondary antibodies (S0001; 1:200; Affinity), respectively. Hematoxylin (Millipore) was incubated with the tissues, and protein expression was analyzed under a CX31‐LV320 microscope (Olympus).
3
Western Blot Analysis of Protein Markers
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Total proteins from tissues specimens and cell lines were extracted using RIPA solution as previously described 20 (link), 21 (link). Briefly, total proteins were separated using SDS-PAGE and transferred to PVDF membrane. After being blocked by non-fat milk, the membranes were incubated with primary antibodies as follows: anti-NSE (Abcam, ab16808, 1:2000), anti-β-catenin (Proteintech, 66009-1-Ig, 1:5000), anti-E-cadherin (Proteintech, 20874-1-AP, 1:5000), and anti-N-cadherin (Affinity, AF4039, 1:1000). Then membranes were incubated with corresponding secondary antibodies and detected using the enhanced chemiluminescence system.
4
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.
5
Cellular Protein Extraction and Western Blot Analysis
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When cells reached 80% confluence, the medium was discarded, and the cells were washed with pre-chilled 1× phosphate-buffered saline (PBS). Then, 320 μL of cell lysis buffer (RIPA buffer supplemented with 3.2 µL PMSF) was added to the cells to extract cellular protein. After a 30-min incubation on ice, the cells were scraped into a 1.5-mL centrifuge tube and subjected to centrifugation at 4°C for 15 min at 12,000 rpm. Protein quantification was performed using a NanoDrop ND-1000. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene fluoride membrane. The membrane was incubated with a primary antibody at 4°C overnight, washed extensively with 1% TBST, and incubated with a secondary antibody conjugated to horseradish peroxidase (1:1000) at room temperature for 3 h. Immunolabelling was visualized using an electrochemiluminescence system. The primary antibodies included anti-N-cadherin (dilution 1:1000, AF4039) and anti-HIF-1α (dilution 1:200, AF1009) antibodies, which were purchased from Affinity (USA), and an anti-GAPDH antibody (dilution 1:2000, AF7021).
6
Protein Expression Analysis by Western Blot
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Total protein was extracted using Radio Immunoprecipitation Assay (RIPA) buffer (P0013B, Beyotime, Jiangsu, China) containing 1 × protease inhibitor cocktail [36 (link)]. Total protein was separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (IPFL00010, Merck, MA, USA). The bands were incubated overnight with primary antibodies at 4°C. Primary antibodies are listed below: cleaved caspase 3 (AF7022, Affinity, CA, USA; 1:1000), caspase 3 (AF6311, Affinity; 1:1000), cleaved poly ADP-ribose polymerase (PARP; AF7023, Affinity; 1:1000), PARP (ab191217, Abcam, Cambridge, UK; 1:1000), cyclin D1 (AF0931, Affinity; 1:1000), cyclin dependant kinase 2 (CDK2; AF6237, Affinity; 1:1000), p27 (AF6324, Affinity; 1:1000), epithelial €-cadherin (AF0131, Affinity; 1:1000), neural (N)-cadherin (AF4039, Affinity; 1:1000), IGF1R (AF6125, Affinity; 1:1000), p-PI3K (AF3241, Affinity; 1:1000), p-AKT (ab38449, Abcam; 1:1000), p-mTOR (AF3308, Affinity; 1:1000), and GAPDH (ab9485, Abcam; 1:1000). The following day, the bands were incubated with goat anti-rabbit IgG (H + L) HRP (S0001, Affinity; 1:5000) for 1 h at room temperature. The results were visualized with ECL reagent (WBKlS0010, Merck), photographed with Gel Imager System, and analyzed using Image J software (1.8.0, National Institutes of Health, USA).
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