Protocol detail

Protein Expression Analysis of Distal Colon

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Distal colonic samples (2–3.5 cm proximal to the anus) were ground and lysed using a total protein extract kit (Keygen). Each sample (30 μg protein) was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Wang et al., 2016 (link)). The membrane was first incubated with primary antibodies against Caspase 3, PCNA, mTORC1, and GCN2 (Cell Signaling) overnight at 4°C and then incubated with the secondary antibody for 2 h at 25°C. The results were visualized via chemiluminescence with EZ-ECL (Biological Industries).

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Zhang H., Hua R., Zhang B., Zhang X., Yang H, & Zhou X. (2018). Serine Alleviates Dextran Sulfate Sodium-Induced Colitis and Regulates the Gut Microbiota in Mice. Frontiers in Microbiology, 9, 3062.

Publication 2018

total protein extract kit primary antibodies against Caspase 3 PCNA mTORC1 EZ-ECL

Antibodies Antibody Anus Biological Caspase 3 Chemiluminescence Colonic Membrane Mtorc1 Nitrocellulose Pcna Protein Sds page

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Abundance/expression levels of Caspase 3, PCNA, mTORC1, and GCN2 proteins

control variables

Distal colonic samples (2–3.5 cm proximal to the anus)
Total protein extract (30 μg protein per sample)
SDS-PAGE separation and nitrocellulose membrane transfer
Primary antibodies against Caspase 3, PCNA, mTORC1, and GCN2 (Cell Signaling)
Overnight incubation of primary antibodies at 4°C
Incubation with secondary antibody for 2 h at 25°C
Visualization via chemiluminescence with EZ-ECL (Biological Industries)

controls

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

Annotations

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