Neuroblastoma cells were lysed in radioimmune precipitation assay buffer containing 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and a mixture of protease inhibitors (Complete, protease inhibitor mixture tablets; Roche Diagnostics) at 4 °C for 30 min. Cell lysates were mixed with SDS-PAGE loading buffer, loaded onto 8–16% gradient Precise Tris-glycine precast gels (Fisher Scientific) and transferred to PVDF membranes (Fisher Scientific). The antibodies used were MYCN (sc-53993, Santa-Cruz Biotechnology, 1:500 dilution), gamma-H2A.X (phospho S139) (ab11174, Abcam, 1:500 dilution), Actin (sc-1616 Santa Cruz Biotechnology, 1:500 dilution), BLM (A300–110A, Bethyl Laboratories, Cambridge Bioscience, 1:1,000 dilution), PKMYT1 (4282S, Cell Signaling, 1:500 dilution), CKS1B (36-6800, Invitrogen 1:500 dilution), SAHH (H00000191-M07A, Abnova, 1:500 dilution), and c-MYC (phospho T58) (ab28842, Abcam, UK, 1:200 dilution). The membranes were then incubated with appropriate HRP-conjugated secondary antibodies: anti-mouse IgG (NXA931, GE Healthcare, Fisher Scientific), anti-rabbit IgG (NA934, GE Healthcare, Fisher Scientific), or anti-goat IgG (sc-2033, Santa Cruz Biotechnology), all at 1:10,000 dilution. Antibody binding was detected by enhanced chemiluminescence (Fisher Scientific).
Sc 2033
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-2033 is a specialized laboratory equipment designed for precise and efficient cell culture applications. The product's core function is to provide a controlled environment for the growth and maintenance of various cell lines. It features advanced temperature regulation, humidity control, and gas flow management systems to ensure optimal conditions for cell cultivation.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor mixture tablet, by Roche (1 mentions)
Ab28842, by Abcam (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ab11174, by Abcam (1 mentions)
Nxa931, by GE Healthcare (1 mentions)
Na934, by GE Healthcare (1 mentions)
H00000191 m07a, by Abnova (1 mentions)
Sc 53993, by Santa Cruz Biotechnology (1 mentions)
Sc 40, by Santa Cruz Biotechnology (1 mentions)
Ab1222, by Merck Group (1 mentions)
Ecl western blotting kit, by Cytiva (1 mentions)
Na931, by GE Healthcare (1 mentions)
Amersham hybond ecl nitrocellulose membranes, by Cytiva (1 mentions)
Na943, by GE Healthcare (1 mentions)
Goat anti actin c 11, by Santa Cruz Biotechnology (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Ab94915, by Santa Cruz Biotechnology (1 mentions)
Ab32249, by Abcam (1 mentions)
5 protocols using sc 2033
1
Neuroblastoma Cell Lysate Analysis
2
Western Blot Analysis of GPATCH3 Protein
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For the Western blot analyses, the tissue extracts, prepared as indicated in the Supplementary Methods section, were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using the Mini-PROTEAN III Gel Electrophoresis System as previously described41 (link). The endogenous GPATCH3 protein present in different tissue extracts was detected using a commercial rabbit polyclonal antibody (HPA032078, Sigma) as the primary antibody, diluted to 1:1000. Horseradish peroxidase-conjugated antibodies against rabbit IgG (#1858415, Pierce) were diluted to 1:1000–1:4000. We used a commercial mouse monoclonal anti-myc antibody (sc-40, Santa Cruz Biotechnology) to detect myc-tagged recombinant proteins, diluted at 1:250. Horse-radish peroxidase-conjugated antibodies against mouse IgG (#32430 ThermoScientific) were diluted to 1:500–1:1000. Chemiluminescence detection and the densitometry for protein band quantification were performed as previously described41 (link). As an additional sample loading control, we detected the endogenous lactate dehydrogenase (LDH) protein in cell extracts using a goat anti-LDH antibody diluted to 1:5000 (AB1222, Chemicon) and an anti-goat IgG horseradish peroxidase-conjugated antibody (sc-2033, Santa Cruz Biotechnology), diluted to 1:2000.
3
Western Blot for ALDH Enzyme Detection
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Cell lysates were prepared in a buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mM sodium pyrophosphate, 1 mM NaF, 1 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM PMSF, 2 μg ml−1 leupeptin and 10 μg ml−1 aprotinin (Sigma Aldrich). A total of 50 μg of cell lysates were separated on SDS–PAGE gel and transferred onto Amersham hybond ECL nitrocellulose membranes (Amersham, Baie d'Urfe, QC, Canada). The membranes were blocked with 5% skimmed milk and then incubated with the indicated antibodies at 4 °C overnight. Appropriate HRP-conjugated secondary antibodies were incubated for 1 h at room temperature. Signals were detected using an ECL Western Blotting Kit (Amersham). The primary and secondary antibodies and the concentrations used were as follows: mouse anti-ALDH1A1 (44/ALDH, 1 : 2000, BD Biosciences, Mississauga, ON, Canada), rabbit anti-ALDH3A1 (B-8, 1 : 500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-actin (C-11, 1 : 1000, Santa Cruz Biotechnology), donkey anti-goat (SC2033, 1 : 3000, Santa Cruz Biotechnology), sheep anti-mouse (NA931, 1 : 3000, GE Healthcare, Mississauga, ON, Canada) and donkey anti-rabbit (NA943, 1 : 3000, GE Healthcare).
4
Western Blotting Protocol for Protein Expression Analysis
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Western blotting was performed using established methodology [13 (link)]. Briefly, cells were washed with PBS and lysed in RIPA buffer. Proteins (20–30 μg) were separated by 10–12% SDS/PAGE and then transferred on an immunoblot PVDF membrane (Bio-Rad, Hercules, CA). After blocking in PBS/Tween (0.1%) with 5% nonfat milk, the membrane was incubated with primary antibodies overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Technology, sc-2033 for anti-goat, and sc-2004 for anti-rabbit, 1:3000) and then developed using ECL solution (Pierce). Primary antibodies used were rabbit anti-β-catenin (Abcam, ab32572, 1:1000), rabbit anti-fibronectin (Santa Cruz Technology, sc-9068, 1:1000), rabbit anti-Wnt3 (Abcam, ab32249, 1:1000), rabbit anti-Wnt7B (Abcam, ab94915, 1:1000), and goat anti-actin (Santa Cruz Technology, sc-1616, 1:3000). For protein expression quantitation, the films were scanned with a CanonScan 9950F scanner and the acquired images were then analyzed using the public domain NIH image program (http://rsb.info.nih.gov/nih-image/ ).
5
Immunohistochemical Analysis of Galectin-1
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Arterial sections were pretreated with retrieval solution (DakoCytomation) at 95 °C for 30 min, followed by incubation with 3% H2O2 for 15 minutes at room temperature to exhaust endogenous peroxidase activity. After blocking with 5% normal donkey serum (017-000-121, Jackson) plus 2% BSA in PBS containing 0.1% Tween-20 (PBST) for 1 h at room temperature, sections were incubated with normal goat IgG (sc-2028, Santa cruz) or goat anti-Gal-1 antibody (AF1245, R&D Systems) diluted in PBST at 4 °C overnight. After 3 washes with PBS, sections were incubated with anti-goat antibody conjugated with horseradish peroxidase (sc-2033, Santa cruz) at room temperature for additional 1 h, followed by PBS wash and antigen detection with 3, 3-diaminobenzidine. Sections were then counter-stained with hematoxylin.
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