Western Blot Analysis of AEG-1 Expression

Western blot is widely used in biochemistry and molecular biology [39 (link), 40 (link)]. In this study, Western blot was used in detecting the expression of AEG-1 protein in different cell lines and different groups. The NSCLC cells were washed with PBS in a 6-well-plate and were lysed in a buffer of 10% glycerol, 5% SDS, 5% 2-mercaptoethanol, 0.2% romophenol blue, 80 mM Tris-HCl (ph 6.8), 5 mM EDTA (ph 8) and 1 mM phenylmethylsulfonyl fluoride. Then, the lysates were centrifuged at 12,000×g for 10 min at 4°C and were boiled for 5 min. Hybond ECL nitrocellulose membranes (GE Healthcare Bio-sciences/Amersham, Diegem, Belgium) were applied to separate the proteins for 2 h at 100 mA. The immunoblot analyses were executed using the following antibodies: AEG-1 (Cell Signaling Technology) and GAPDH (Vazyme Biotech Co., Ltd). The image pro plus 6.0 was used to analyze the IOD value.

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Zhang Y., Li Z.Y., Hou X.X., Wang X., Luo Y.H., Ying Y.P, & Chen G. (2017). Clinical significance and effect of AEG-1 on the proliferation, invasion, and migration of NSCLC: a study based on immunohistochemistry, TCGA, bioinformatics, in vitro and in vivo verification. Oncotarget, 8(10), 16531-16552.

Publication 2017

Hybond ECL nitrocellulose membranes AEG-1 GAPDH

Antibodies Buffer Cell lines Cells Edta Gapdh Glycerol Immunoblot analyses Membranes Mercaptoethanol Nitrocellulose Nsclc Phenylmethylsulfonyl fluoride Proteins Tris Western blot

Corresponding Organization : Guangxi Medical University

Other organizations : Union Hospital, Jilin University

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Variable analysis

independent variables

None specified

dependent variables

Expression of AEG-1 protein in different cell lines and different groups

control variables

Treatment of NSCLC cells (washed with PBS in a 6-well-plate)
Lysis buffer (10% glycerol, 5% SDS, 5% 2-mercaptoethanol, 0.2% bromophenol blue, 80 mM Tris-HCl (pH 6.8), 5 mM EDTA (pH 8), and 1 mM phenylmethylsulfonyl fluoride)
Centrifugation (12,000×g for 10 min at 4°C)
Boiling of lysates (5 min)
Protein separation using Hybond ECL nitrocellulose membranes (GE Healthcare Bio-sciences/Amersham, Diegem, Belgium) for 2 h at 100 mA
Antibodies used for immunoblot analyses (AEG-1 and GAPDH)
Image analysis software (Image Pro Plus 6.0)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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