Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation
January 18th, 2019
Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.
This protocol allows researchers to obtain distinct fractions of a cell without the use of specialized equipment, expensive reagents or complex techniques.The primary advantage of this protocol is the simplicity of the techniques which make it modifiable and easy to perform.Demonstrating the procedure will be Matt Deragon, Rob Haluska and Alexa Hodges.Three graduate students from my laboratory.To begin centrifuge the prepared cell suspension to create a pellet.Remove the supernatant and re-suspend the pellet in room temperature PBS.Centrifuge the cell suspension at 400 xg for ten minutes.Then remove the supernatant and re-suspend the cell pellet in ice cold buffer.Prepare buffer A solution according to the text protocol.Centrifuge the cell suspension at 400 xg for ten minutes and remove the supernatant.Re-suspend the pellet in buffer A solution to a final concentration of 200 million cells per ml, and pipette gently to break up any clumps.After this incubate the cell suspension on an end over end rotator at 4
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Overview
This article presents a protocol for isolating the plasma membrane, cytoplasm, and mitochondria of U937 cells without high-speed centrifugation. The technique allows for the purification of subcellular fractions for protein localization studies via immunoblotting.
Key Study Components
Area of Science
- Cell Biology
- Biochemistry
- Neuroscience
Background
- U937 cells are a model for studying human monocyte function.
- Isolation of subcellular components is crucial for understanding cellular processes.
- Traditional methods often involve high-speed centrifugation, which can be harsh on cells.
- This protocol aims to provide a gentler alternative for subcellular fractionation.
Purpose of Study
- To develop a protocol for isolating cell components without high-speed centrifugation.
- To enable subsequent analysis of protein localization.
- To enhance the understanding of cellular mechanisms in U937 cells.
Methods Used
- Isolation of plasma membrane, cytoplasm, and mitochondria.
- Use of specific reagents to facilitate cell fractionation.
- Immunoblotting for protein localization analysis.
- Comparison of results with traditional methods.
Main Results
- Successful isolation of subcellular fractions without high-speed centrifugation.
- Clear visualization of protein localization via immunoblotting.
- Demonstrated efficiency and gentleness of the new protocol.
- Potential for broader applications in cellular biology research.
Conclusions
- The new protocol is effective for isolating U937 cell components.
- It provides a viable alternative to traditional methods.
- Future studies can utilize this technique for various cellular analyses.
