May 14, 2020
Western blot analysis and immunoprecipitation assay
- 1Laboratorio Cellule Staminali Post Natali e Terapie Cellulari, IRCCS Istituto Gaslini, Genova, Italy
External link: https://doi.org/10.1371/journal.pone.0244069
Protocol Citation: Marzia Ognibene 2020. Western blot analysis and immunoprecipitation assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bgecjtaw
Manuscript citation:
Ognibene M, Pezzolo A (2020) Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma. PLoS ONE 15(12): e0244069. doi: 10.1371/journal.pone.0244069
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: May 14, 2020
Last Modified: May 14, 2020
Protocol Integer ID: 37028
Cells are washed in cold PBS and lysed on ice in a buffer containing 1.6 mM NaH2PO4, 8.6 mM Na2HPO4, 1% Triton X-100, 0.1% SDS; 0.1% NaCl, 0.5% NaDoc, 2 mM AEBSF, 20 mg/mL each of aprotinin and leupeptin.
Cell lysates are centrifuged in a microfuge at maximum speed for 10 min to eliminate debris.
Protein content is evaluated by bicinchoninic acid assay (BCA Protein assay kit by Thermo Fisher) in a spectrophotometer.
Lysates (100 µg each) are subjected to SDS-PAGE electrophoresis and transferred to PVDF membrane.
For the IMMUNOPRECIPITATION ASSAY, cells are lysed in a buffer containing 20 mM Tris-HCl, 1% Triton X-100, 10% Glycerol, 2 mM AEBSF, 20 mg/mL each of aprotinin and leupeptin.
Lysates (1000 µg) are incubated overnight at 4˚C with the desired antibody under constant rotation.
Gammabind G-Sepharose (40 µl) is added to each sample and let rotate for 1 hour and 30 min.
Samples are washed three times with the lysis buffer in a microfuge at 4300 g for 1 min.
After eliminating the last supernatant, samples are eluted in 40 µl of 1x Laemmli’s buffer, subjected to SDS-PAGE and transferred to PVDF membrane.
PVDF membrane is incubated in blocking buffer (4% nonfat milk in T-TBS -Tris Buffer Solution 1x with 0,05%Tween) for 2 hr at room temperature.
Membrane is incubated in primary antibody diluted in 0,5%BSA in T-TBS, overnight at 4°C.
Membrane is washed in T-TBS, 5 times, 5 min each.
Membrane is incubated in HRP-conjugated secondary antibody diluted in 0,5%BSA in T-TBS, 2 hr, at room temperature.
Membrane is washed in T-TBS, 5 times, 5 min each.
Proteins are visualized by enhanced chemiluminescence (ECL) detection, through the Bio-Rad ChemiDoc instrument. Signal intensity was measured by densitometry using the Bio-Rad Image Lab 6.0 software.