Protein extracts were resolved on 12% and 10% SDS–PAGE gels, transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and blocked in 5% skim milk. The following primary antibodies were used according to the manufacturer's instructions: anti-CD133 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ADAM17 (ab cam), NICD (Cell Signaling Technology, Beverly, MA, USA), MTSS1 (Santa Cruz Biotechnology), HES1 (Santa Cruz Biotechnology) and β-actin (Sigma-Aldrich Co.). After incubation with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences, Cardiff, UK), specific protein bands were visualized using enhanced chemiluminescence (Amersham Biosciences). The density of each band was measured using the TINA software.
Anti cd133
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-CD133 is a laboratory reagent that targets the CD133 antigen, a cell surface marker. It is commonly used in research applications to identify and isolate specific cell populations expressing the CD133 protein. The core function of Anti-CD133 is to enable the detection and separation of CD133-positive cells, which can provide insights into various biological processes and disease states. No further interpretation or extrapolation on the intended use of this product is provided.
Automatically generated - may contain errors
Lab products found in correlation
Anti nestin, by Merck Group (3 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Adam17, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse or anti rabbit secondary antibodies, by Cytiva (1 mentions)
β actin, by Merck Group (1 mentions)
Anti β 3 tubulin, by Cell Signaling Technology (1 mentions)
Anti mbp, by Santa Cruz Biotechnology (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Anti p smad2 3, by Cell Signaling Technology (1 mentions)
Anti smad2 3, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Santa Cruz Biotechnology (1 mentions)
Anti p egfr tyr1068, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti c jun, by Santa Cruz Biotechnology (1 mentions)
Anti fos, by Santa Cruz Biotechnology (1 mentions)
Anti smad4, by Santa Cruz Biotechnology (1 mentions)
Anti p p44 42, by Cell Signaling Technology (1 mentions)
Anti p c jun, by Cell Signaling Technology (1 mentions)
6 protocols using anti cd133
1
Protein Expression Analysis by Western Blot
2
Stemness and Differentiation Markers
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To evaluate the expression of stemness and differentiation markers, the following antibodies were used: anti-CD133 (1:50, Santa Cruz Biotechnology, Dallas, TX, USA); anti-nestin (1:50, Millipore, Burlington, MA, USA,); anti-GFAP (1:200, DakoCytomation, Glostrup, Denmark); anti-βIII Tubulin (1:100, Cell Signaling, Danvers, MA, USA); anti-MBP (1:50, Santa Cruz Biotechnology, Dallas, TX, USA). Each marker was analyzed in a separate set of experiments and with at least two replicates.
3
Western Blot Analysis of Cell Signaling
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Western blotting was performed as described previously [20 (link),21 (link)]. The following antibodies were used in this study: anti-SMAD4 (sc-7154 or sc-7966), anti-E-cadherin (sc-8426), anti-vimentin (sc-7557), anti-CD133 (sc-8304), anti-CD44 (sc-18849), anti-Sp1(sc-14027), anti-c-Jun (sc-1694), anti-Fos (sc-52), anti-Fast-1 (sc-377358), anti-Hes1 (sc-25392), anti-GAPDH (sc-32233; Santa Cruz Biotechnology, Inc.), anti-p-Akt (#4060), anti-Akt (#4691), anti-p-p44/42 (#9101),anti-p44/42 (#4695), anti-Pten (#9272), anti-NF-κB (#4764S), anti- EGFR (#4267), anti-p-EGFR tyr 992 (#2235), anti-p-EGFR tyr 1068 (#3777), anti-Smad2/3 (#5339), anti-p-Smad2/3 (#3101), anti-p-c-Jun (#2361; Cell Signaling Technology, Inc.), anti-Nestin (N5413), mouse anti-β-actin (Sigma- Aldrich Co.), anti-CD133/1 (AC133, Miltenyi Biotec.) and anti-TGF-β1 (ab9758, Abcam, Plc.).
4
Immunoblotting Analysis of Stem Cell Markers
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Immunoblotting were performed as previously described45 (link). The protein content was determined using Bradford Reagent (#5000006; Bio‐Rad) and the antibodies used were purchased from Cell Signaling: anti-caspase3 (#9662) and anti-pH2A.X (#9718); Millipore: anti-nestin (#MAB5326), anti-CD133 (#MAB4399), and anti-SOX2 (#AB5603); or from Santa Cruz Biotechnology (Dallas, USA): anti-GFAP (#sc-6171-R). ImageJ bundled with Java 1.8.0_172 (U. S. National Institutes of Health, Maryland, USA) was used to normalize intensity levels according to loading controls expression blotted with anti‐GAPDH (#AM4300; ThermoFisher Scientific) or anti‐β‐actin (#sc-69879; Santa Cruz Biotechnology). Original immunoblots are represented in Suppl. Figure S11.
5
CD133 Protein Expression Analysis in Cells
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Sorted cells were collected and nuclear protein was extracted using Nuclear Extraction Protocol (Thermo Fisher Scientific, Shanghai, China). Total amounts of total protein (30 µg) were separated using SDS-PAGE and transferred to a poly-vinylidene fluoride membrane. The membranes were blocked in 5% nonfat milk for 1 hour and incubated at 4°C overnight with primary antibodies: anti-CD133 (1:1,000, sc-101199, Santa Cruz), anti-histone H3 (1:5,000, Abcam, Shanghai, China). Signals were visualized after incubation with horseradish peroxidase-conjugated secondary antibody.
6
Glioblastoma Stem Cells Identification
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After cultured in the serum-free medium for 2 weeks, the primary spheres formed and reached 100–200 cells each, the cells remaining in the flasks were the non-glioblastoma stem cells (non-GSCs). The cultures were harvested by mechanical centrifugation, trypsinized into single-cell suspensions and plated into a 96-well plate for the subsphere-forming assay by limiting dilution as described previously [26] (link), [27] (link), [28] (link). Meanwhile, the monolayer cells remaining in the flasks after harvesting the spheres were transferred into medium without growth factors but permissive for differentiation. Spheres and differentiated cells were cultured in pre-coated chamber slides and fixed, and then were stained with the antibodies as follows: anti-Nestin (rabbit, monoclonal, IgG1, 1∶100, Santa Cruz Biotechnology, USA), anti-CD133 (mouse, monoclonal, IgG1, 1∶100, Santa Cruz Biotechnology, USA), anti-GFAP (rabbit, polyclonal, 1∶100, Abcam, MA, USA), anti-beta-tubulinIII (mouse, monoclonal, IgG1, 1∶100, Santa Cruz Biotechnology, USA). The primary antibodies were detected with cy3-conjugated anti-mouse and FITC-conjugated anti-rabbit IgG antibodies (1∶200, Beyotime Institute of Biotechnology, Jiangsu, China). Cells were also counterstained with DAPI to identify all nuclei.
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