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RNeasy Mini Handbook 10/2019
87
Appendix F: Acetone Precipitation of Protein from
Buffer RLT Lysates
This protocol is designed for acetone precipitation of protein from cell lysates prepared using
Buffer RLT. The precipitated, denatured protein is suitable for applications such as SDS-PAGE,
western blotting, and 2D gel electrophoresis.
Equipment and reagents to be supplied by user*
Ice
Benchtop centrifuge Acetone
Optional: Ethanol
Buffer for downstream application (e.g., loading buffer for SDS-PAGE gel)
Important point before starting
Important: DO NOT use trichloroacetic acid (TCA) to precipitate protein from Buffer RLT
lysates.
This buffer contains guanidine thiocyanate, which can form highly reactive compounds
when combined with acidic solutions.
Procedure
1. Prepare cell lysate and centrifuge it through an RNeasy spin column, as described in the
protocols in this handbook.
2. Add 4 volumes of ice-cold acetone to the flow-through from the RNeasy spin column.
3. Incubate for 30 min on ice or at −30 to −15°C.
*
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more
information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
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