Is there any difference between the protein extraction protocol from frozen cells and cultured cells?

Hello Maria Clara Sampaio

No.

But regarding the frozen cells, you need to snap freeze the cells on dry ice and store the cell pellet at -80 degree C until further use.

When required, resuspend the cell pellet in the desired volume of an appropriate lysis buffer containing protease inhibitors and continue with the subsequent steps of the protein extraction protocol.

Best.

Aarya Chitransh

Yes, there can be differences in the protein extraction protocol when working with frozen cells compared to cultured cells. The choice of method depends on the specific requirements of your experiment and the downstream applications for the extracted proteins. Here are some considerations for each scenario:

Protein Extraction from Frozen Cells:

Cell Lysis:Frozen cells can be more resistant to lysis compared to cultured cells. Mechanical disruption methods such as homogenization or grinding may be necessary to effectively lyse frozen cells. Additionally, freeze-thaw cycles can help break cell membranes and facilitate lysis.

Buffer Composition:The composition of the lysis buffer may need to be adjusted to account for differences in cell integrity after freezing. Including protease inhibitors and phosphatase inhibitors in the lysis buffer is crucial to prevent protein degradation.

Temperature Control:Work on ice or in a cold room during the extraction process to minimize protein degradation. Keep all buffers and equipment chilled to maintain protein stability.

Centrifugation:Depending on the freezing method and cell type, there may be changes in cell morphology that affect the efficiency of centrifugation. Optimize centrifugation conditions to effectively separate the cellular components.

Protein Extraction from Cultured Cells:

Cell Harvesting:For cultured cells, harvesting is usually straightforward, involving trypsinization or scraping. Properly detach cells from the culture vessel to ensure a representative sample.

Lysis Buffer:Lysis buffers for cultured cells should include detergents to disrupt the cell membrane. Triton X-100, NP-40, or SDS are commonly used detergents. The buffer composition may also include salts and protease inhibitors.

Sonication:In some cases, sonication may be necessary for efficient disruption of cultured cells, especially if you're working with adherent cells that form a tight monolayer.

Centrifugation:Centrifugation steps are crucial to separate soluble proteins from cell debris and organelles. Optimize centrifugation speeds and durations based on the specific requirements of your experiment.

General Tips:

Protease and Phosphatase Inhibitors:Include protease and phosphatase inhibitors in your lysis buffer regardless of the source of cells. This helps preserve the integrity of proteins during extraction.

Sample Storage:If you're working with frozen cells, ensure proper storage conditions to maintain protein stability. Flash-freeze cells in liquid nitrogen or dry ice and store them at -80°C.

Pilot Experiments:Conduct pilot experiments to optimize the protein extraction protocol for your specific cell type, whether frozen or cultured.

Always follow established protocols, and consider the nature of your cells and the downstream applications for the extracted proteins when adapting or developing a protein extraction protocol.

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