Due to change of plans, I need to extract protein from my samples which are kidney, heart and liver of mice. I’ve fixed them in formalin and then did a series of various concentration of sucrose solution and now it’s stored in OCT. Is there any way that I could extract proteins from my samples to run LCMS?
Preetha Rajendiran 1. Use xylene washes followed by graded ethanol washes to remove paraffin and rehydrate the tissue
2. Incubate in a retrieval buffer (e.g., Tris-EDTA pH 9 or citrate buffer pH 6) at 95–100°C for 20–60 minutes.
3. Use a strong lysis buffer (e.g., RIPA buffer with SDS and protease inhibitors).Homogenize using bead mill, sonication, or mechanical disruption. Some protocols use commercial kits specifically designed for FFPE or PFA-fixed tissue protein extraction.
4. Use BCA or Bradford assay, but be cautious as detergents and retrieval buffers can interfere
5. For Western blot, expect higher molecular weight smearing due to incomplete cross-link reversal. For Mass Spectrometry, additional digestion steps (trypsin) and detergents may be needed. For Enzyme activity assays are usually not possible, as fixation denatures proteins.
Protein extraction from fixed mouse organs can be challenging due to the cross-linking effects of fixation. However, several methods have been developed to optimize protein recovery:
1. TRIzol®-Based Extraction: TRIzol® can be used for simultaneous isolation of RNA, DNA, and proteins. Modifications to the standard protocol, such as using Urea:SDS solubilization and sonication, improve protein recovery. This method has been validated for tissues stored long-term in TRIzol®-chloroform fractions.
2. Buffer Optimization: For fresh or fixed tissues, using phosphate-buffered saline (PBS) supplemented with protease inhibitors and hand-homogenization has been shown to yield high protein recovery. This approach is particularly effective for mouse tissues like ovaries.
3. SDS Lysis Buffer: Sodium dodecyl sulfate (SDS) is a cost-effective lysis buffer for protein extraction. For mouse liver tissues, a 1.5% SDS solution has been found to maximize protein yield.
4. Membrane Protein Isolation: For isolating membrane proteins, methods like the peptidisc approach can be used. This technique captures membrane proteins in a water-soluble form, enabling downstream analysis.
Each method has specific applications depending on the type of proteins and the downstream analysis required.
To extract protein from a fixed mouse organ, you'll need to reverse the effects of the fixative and then lyse the cells. A common approach involves deparaffinization, rehydration, and protein extraction using a buffer with a detergent like SDS, followed by heat treatment. The extracted proteins can then be analyzed using techniques like Western blotting.
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