Primary antibodies against β-actin, FAK (Focal adhesion kinase), p-397Tyr-FAK, IKKα (I-kappaB kinase α), IKKβ (I-kappaB kinase β), β-catenin, total-p53, p21, and MnSOD were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA). Primary antibodies against UCP2, NF-κB p65, p-536Ser-NF-κB p65, S6 ribosomal protein and cleaved caspase 3, were purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibodies against wild type p53 were purchased from MilliporeSigma (Burlington, MA, USA). On a 10% SDS–PAGE gel, 20 μg total protein was electrophoresed, transferred onto a polyvinylidene fluoride membranes, blocked, incubated with a primary antibody and then with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Immunoreactive bands were visualized using a chemiluminescence solution (Genesee Scientific, El Cajon, CA, USA). Experiments were repeated three times. β-actin was employed as an endogenous control. The pixel densities of the protein bands were quantified using ImageJ.
Chemiluminescence solution
Manufactured by Genesee Scientific
Sourced in United States
Chemiluminescence solution is a reagent used in various analytical techniques. It produces light emission as a result of a chemical reaction, which can be detected and measured. The core function of this solution is to enable chemiluminescence-based detection and quantification of target analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Total p53, by Santa Cruz Biotechnology (1 mentions)
Mnsod, by Santa Cruz Biotechnology (1 mentions)
S6 ribosomal protein, by Cell Signaling Technology (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
2 protocols using chemiluminescence solution
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Proteins
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Protein samples (20 µg) of whole-cell lysate were separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked and incubated with a primary antibody, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA). Immunoreactive bands were visualized using a chemiluminescence solution (Genesee Scientific, El Cajon, CA, USA). Experiments were repeated three times. β-actin was used as the loading control for cell lysis. The protein bands were quantified using the ImageJ software.
For analysis of culture supernatants, the culture medium was centrifuged and filtrated with a 0.22 µm filter to remove cells. Supernatant samples (30 µl) were combined with 4 x loading buffer (10 µl), denatured, separated by SDS-PAGE, and analyzed as described above. Ponceau S staining of the PVDF membrane was used as the loading control for electrophoresis of the culture medium.
For analysis of culture supernatants, the culture medium was centrifuged and filtrated with a 0.22 µm filter to remove cells. Supernatant samples (30 µl) were combined with 4 x loading buffer (10 µl), denatured, separated by SDS-PAGE, and analyzed as described above. Ponceau S staining of the PVDF membrane was used as the loading control for electrophoresis of the culture medium.
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