Semi-denaturing Agarose Gel Electrophoresis

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was conducted according to the published protocol^{36 (link)}. Crude cell lysis was resuspended in 1×sample buffer (0.5×TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad, USA). After electrophoresis in the running buffer (1×TBE and 0.1% SDS) for 35 min with a constant voltage of 100 V at 4 °C, the proteins were transferred to Immobilon membrane (Millipore, USA) for immunoblotting and the following procedures are same as the Western-blot assay.

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Zhuang T., Yi X., Chen J., Kang P., Chen X., Chen J., Cui T., Chang Y., Ye Z., Ni Q., Wang Y., Du P., Li B., Liu L., Jian Z., Li K., Gao T., Li S, & Li C. (2020). Intracellular virus sensor MDA5 exacerbates vitiligo by inducing the secretion of chemokines in keratinocytes under virus invasion. Cell Death & Disease, 11(6), 453.

Publication 2020

vertical 1.5% agarose gel Immobilon membrane

Agarose Agarose gel electrophoresis Bromophenol blue Buffer Cell Detergent Electrophoresis Glycerol Immobilon Membrane Proteins Western blot assay

Corresponding Organization :

Other organizations : Air Force Medical University, Xijing Hospital

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Variable analysis

independent variables

Detergent concentration (SDS) in the sample buffer

dependent variables

Migration of proteins on the agarose gel
Protein transfer to the Immobilon membrane

control variables

Voltage (100 V)
Duration of electrophoresis (35 min)
Temperature (4 °C)
TBE buffer concentration (0.5× in sample buffer, 1× in running buffer)
Glycerol concentration (10%) in sample buffer
Bromophenol blue concentration (0.0025%) in sample buffer

Annotations

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