TRAMP C2H cells were plated at 5 x105 cells/10 cm tissue culture dish in 10 mL media. Cells were left to adhere overnight, rinsed with PBS and then fresh media was added. Cells were then transfected with miR-30e* inhibitor. Twenty-four hours later cells were rinsed with PBS twice and harvested. Protein lysates were generated using cell lysis buffer #6 (R&D Systems Minneapolis, MN). The ELISA was purchased from R&D Systems and run according to the provided protocol. One hundred and twenty-five μg of protein was used per well and all samples were run in duplicate.
Cell lysis buffer 6
Manufactured by R&D Systems
Cell lysis buffer #6 is a solution designed to facilitate the disruption and extraction of cellular contents. It is intended for use in a variety of cell biology and biochemical applications that require the isolation of cellular components, such as proteins, nucleic acids, or organelles. The buffer's formulation and properties are optimized to effectively lyse cells while preserving the integrity of the target analytes.
Automatically generated - may contain errors
2 protocols using cell lysis buffer 6
1
Measuring miR-30e* Inhibitor Effects
2
Western Blot Analysis of Protein Expression
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Cells were lysed (cell lysis buffer 6, R&D Systems) and denatured in 1× Laemmli sample buffer (Bio-Rad) containing 2-mercaptoethanol (Sigma-Aldrich). Proteins were separated on Criterion Tris-Glycine eXtended gels (Bio-rad), transferred to PVDF membranes, and incubated with primary and secondary antibodies (Supplementary Table 3 ). Proteins were visualised using chemiluminescence on an Azure C600 (Azure Biosystems). Quantification was performed using LI-COR Image Studio Lite software version 5.2. Vinculin and β-actin were used to normalise for protein input. Immunoblots were performed on samples from at least three biological repeat experiments.
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