Aug 09, 2023
Preparing whole cell samples for immunoblot analysis
- 1Lazarou Lab, WEHI
Protocol Citation: Louise Uoselis 2023. Preparing whole cell samples for immunoblot analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdj4dlmk/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: 86214
Keywords: ASAPCRN
Abstract
Protocol for preparation of HeLa cell lysates for immunoblot analysis.
Add an appropriate volume of 1x LDS Sample Buffer (ThermoFisher) to each sample.
Boil each sample at 99 °C with shaking at maximum speed for 00:10:00
10m
Allow all samples to cool to Room temperature , and quickly centrifuge the samples to collect all liquid in the bottom of the tube. Vortex each sample for ~3 seconds to ensure homogeneity.
Measure the concentration of each sample spectroscopically, blanking with the 1x LDS sample buffer and using an A280 measurement. Dilute samples with 1x LDS Sample buffer to a concentration of < 7 mg/mL if required.
Aliquot out the desired amount of each sample into a separate tube, and add 1x LDS Sample Buffer to each sample to make all samples in the same gel a standard volume.
Either freeze samples at -20 °C until required, or load directly onto a SDS-PAGE gel.