How to improve the protocol for isolation of stress granules from mammalian cells?

23 April 2024 2 2K Report

Hello all, I am currently in the process of isolating stress granules from mammalian cells transfected with GFP-tagged FUS mutant.

I could see the stress granules clearly before and after cell lysis. However, I could not see stress granules after the centrifugation by fluorescence microscope.

I followed this protocol: https://www.sciencedirect.com/science/article/abs/pii/S1046202316303371?via%3Dihub

In brief:

(1) 2 plates (15 cm dish) of cells were scraped, spun down, and re-suspended in 400 µL of lysis buffer. Cells were lysed through sonication (2 seconds followed by 2 seconds break, 5 cycles) on ice.

Stress granule lysis buffer—50 mM Tris HCl pH 7.4, 100 mM Potassium acetate, 2 mM Magnesium acetate, 0.5 mM DTT, 50 μg/mL Heparin, 0.5% NP40, 1:5000 Antifoam B, 1 complete mini EDTA free protease inhibitor tablet 50/mL of lysis buffer. *Add RnaseIN 0.1 U/μL right before lysis.

(2) After lysis, spin at 1000g, 5 min at 4 °C to pellet cell debris. The supernatant was transferred to 1.5 mL microcentrifuge tube.

(3) The supernatant was spun with 18,000g, 20 min at 4 °C. The supernatant was discarded.

(4) The pellet was re-suspended in 400 µL of stress granule lysis buffer, spin 18,000g, 20 min at 4 °C. The supernatant was discarded.

(5) The pellet was re-suspended again with lower volume of lysis buffer (120 µL) and spun down with 850g, 2 min at 4 °C. Supernatant was transferred to a new 1.5 mL microcentrifuge tube. The supernatant represents the mammalian stress granule core enriched fraction.

I noticed the debris was not totally removed at step (2). Could it affect the yield of stress granules? Should I use higher speed or longer time of centrifugation to remove the debris? Or the centrifugation of step (3) and (4) need to be optimized?

Your expertise and guidance in this area would be greatly appreciated. Thank you in advance for your assistance.

Kais Khudhair al Hadrawi

Isolating stress granules from mammalian cells is crucial for studying various cellular processes. Here are some ways to improve the protocol for isolating stress granules:

Optimization of Cell Lysis: Ensure efficient cell lysis to release stress granules. Adjust the lysis buffer composition, including detergents (such as Triton X-100 or NP-40) and protease inhibitors, to maintain stress granule integrity.

Centrifugation Parameters: Optimize centrifugation parameters (speed, time, and temperature) to pellet stress granules effectively while minimizing contamination with other cellular components.

RNA Stabilization: Incorporate RNase inhibitors in the lysis buffer to prevent RNA degradation during stress granule isolation, as RNA is a crucial component of stress granules.

Antibody-Based Isolation: Utilize specific antibodies against stress granule markers (such as TIA-1, G3BP, or eIF4E) for immunoprecipitation-based isolation. This method ensures high specificity and purity of isolated stress granules.

Cross-linking Agents: Use cross-linking agents like formaldehyde to stabilize protein-protein and protein-RNA interactions within stress granules before cell lysis. This step can enhance the preservation of stress granule integrity during isolation.

Gradient Centrifugation: Employ density gradient centrifugation to separate stress granules from other cellular components based on their buoyant density. This method can improve the purity of isolated stress granules.

Cryo-Electron Microscopy (Cryo-EM): Combine stress granule isolation with cryo-EM techniques to visualize stress granule structure at high resolution. Cryo-EM provides detailed insights into stress granule morphology and composition.

Functional Assays: Develop functional assays to validate the biological activity of isolated stress granules, such as RNA granule formation assays or stress-induced translational repression assays.

Validation of Isolated Stress Granules: Confirm the presence of stress granules in isolated fractions using microscopy, Western blotting, or RNA analysis. This step ensures the specificity of isolated stress granules.

Iterative Optimization: Continuously refine the isolation protocol based on experimental outcomes and literature insights to enhance the efficiency and reproducibility of stress granule isolation.

Harem Muhamad Amin

You can follow my Co-IP protocols, especially for cross-linkings and lysis buffers, in the following articles:

doi: 10.1002/pro.4847

doi: 10.3390/ijms242216032

You will need some little modifications. I can provide you necessary details via this email: [email protected]

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