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Gradient minigels

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4–12% gradient minigels are laboratory equipment designed for electrophoresis separation of proteins. They provide a gradient of polyacrylamide concentrations, allowing for effective separation of a wide range of protein molecular weights in a single gel.

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NuPAGE 4–12% Bis-Tris gradient minigels

2 protocols using gradient minigels

1

Western Blotting Analysis of Tumor Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols
Tumor lysates described in RPPA section above were also subjected to gel electrophoresis using 25μg protein per lane of pre-cast 4–12% gradient minigels (Novex, Life Technologies), followed by transfer onto Nitrocellulose membrane (Novex, Life Technologies), blocking with 5% dry milk in TBST for 1h, shaking with supplier-recommended concentration of primary antibodies (all from Cell Signaling Technology) at room-temperature for 4h or at 4° C overnight, and developing with secondary antibodies that were labeled for detection with Odyssey system. Labeled molecular weight marker was also loaded on the gels. Protein bands were quantified using Odyssey features with scan times set to exclude overexposure. For representative images shown in Fig. 2b and Supplementary Fig. 2, at least three independent experiments were performed with similar images obtained.
Welte T., Kim I.S., Tian L., Gao X., Wang H., Li J., Holdman X.B., Herschkowitz J.I., Pond A., Xie G., Kurley S., Nguyen T., Liao L., Dobrolecki L.E., Mo Q., Edwards D.P., Huang S., Xin L., Xu J., Li Y., Lewis M.T., Wang T., Westbrook T.F., Rosen J.M, & Zhang X.H. (2016). Oncogenic mTOR signaling recruits myeloid-derived suppressor cells to promote tumor initiation. Nature cell biology, 18(6), 632-644.
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2

Western Blotting Analysis of Tumor Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols
Tumor lysates described in RPPA section above were also subjected to gel electrophoresis using 25μg protein per lane of pre-cast 4–12% gradient minigels (Novex, Life Technologies), followed by transfer onto Nitrocellulose membrane (Novex, Life Technologies), blocking with 5% dry milk in TBST for 1h, shaking with supplier-recommended concentration of primary antibodies (all from Cell Signaling Technology) at room-temperature for 4h or at 4° C overnight, and developing with secondary antibodies that were labeled for detection with Odyssey system. Labeled molecular weight marker was also loaded on the gels. Protein bands were quantified using Odyssey features with scan times set to exclude overexposure. For representative images shown in Fig. 2b and Supplementary Fig. 2, at least three independent experiments were performed with similar images obtained.
Welte T., Kim I.S., Tian L., Gao X., Wang H., Li J., Holdman X.B., Herschkowitz J.I., Pond A., Xie G., Kurley S., Nguyen T., Liao L., Dobrolecki L.E., Mo Q., Edwards D.P., Huang S., Xin L., Xu J., Li Y., Lewis M.T., Wang T., Westbrook T.F., Rosen J.M, & Zhang X.H. (2016). Oncogenic mTOR signaling recruits myeloid-derived suppressor cells to promote tumor initiation. Nature cell biology, 18(6), 632-644.
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