The cytosol-mitochondria fractionation assay was performed using a Mitochondria/Cytosol Fractionation Kit (Millipore) as previously described46 (link), 47 (link). Briefly, NHLFs were transfected for 30 h with either non-targeting control siRNA or ISG15-specific siRNA and then transfected with EMCV-RNA or RABVLe for 16 h. Cells were homogenized in an isotonic buffer using a Dounce homogenizer and the lysates were centrifuged at 600 ×g to pellet the nuclei and unbroken cells. The supernatant was further centrifuged at 10,000 ×g at 4°C for 30 min to separate the cytosolic (supernatant) and mitochondrial (pellet) fractions. The protein concentration of both fractions was determined by a bicinchoninic acid (BCA) assay (Pierce), and equal amounts of proteins were analyzed by immunoblotting. Anti-α-tubulin and anti-MAVS immunoblotting served as markers for the cytosolic and mitochondrial fractions, respectively.
Protocol detail
Cytosol-Mitochondria Fractionation Assay
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Assay
Bicinchoninic acid
Buffer
Cells
Cytosolic
Emcv
Fractionation
Mavs
Mitochondrial
Nuclei
Proteins
Sirna
α tubulin
Corresponding Organization :
Other organizations : Cleveland Clinic Florida, University of Chicago, Harvard University
Top 5 similar protocols
Variable analysis
independent variables
- SiRNA transfection (non-targeting control siRNA or ISG15-specific siRNA)
- Virus transfection (EMCV-RNA or RABV_Le)
- Cytosolic and mitochondrial fractions
- Protein concentration in cytosolic and mitochondrial fractions
- Presence of α-tubulin (cytosolic marker) and MAVS (mitochondrial marker)
- Homogenization in isotonic buffer
- Centrifugation conditions (600 × g, 10,000 × g, 4°C, 30 min)
- Protein quantification method (BCA assay)
- Positive control: Anti-α-tubulin immunoblotting for cytosolic fraction
- Positive control: Anti-MAVS immunoblotting for mitochondrial fraction
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!