Aug 09, 2023
Preparing mitochondrial samples for immunoblot analysis
- 1Lazarou Lab, WEHI
Protocol Citation: Louise Uoselis 2023. Preparing mitochondrial samples for immunoblot analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj38enlk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86215
Keywords: ASAPCRN, mitochondrial samples for immunoblot analysis protocol, mitochondrial samples for immunoblot analysis, preparation of mitochondrial sample, preparing mitochondrial sample, mitochondrial sample, immunoblot analysis, immunoblot analysis protocol
Abstract
Protocol for preparation of mitochondrial samples for immunoblot analysis.
Thaw mitochondrial stocks on ice, and aliquot out the desired amount of mitochondria.
Centrifuge each aliquot for 00:10:00 10000 x g, 4°C
10m
Carefully aspirate the supernatant from each sample.
Add a volume of 1x SDS sample buffer (5% w/v SDS, 10% v/v glycerol, 100 mM DTT, 50 mM Tris-Cl pH 6.8) equal to the amount of mitochondria (in ug) to each sample. Eg. If each sample contains 20 µg of mitochondria, add20 µL of 1x SDS sample buffer.
Vortex samples for ~5 seconds to mix, and then boil at 99 °C with shaking at max speed for 00:10:00 .
10m
Allow samples to cool to room temperature, quickly centrifuge to collect liquid to the bottom of the tube, and vortex for ~3 seconds to ensure the samples are homogenous.
Samples can now be directly loaded onto an SDS-PAGE gel, or stored at -20 °C for later use.