Protein Lysate Extraction: A Technique to Lyse Organoids to Collect Cellular Proteins

Published: April 30, 2023

Abstract

Source: Crowell, P. D. et al. Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture. J. Vis. Exp. (2019)

In this video, we demonstrate the extraction of protein lysates from prostate organoids cultured in a basement membrane matrix ring. Analysis of the extracted protein fractions can help study the lineage marker expression in the organoids.

Protocol

1. Extracting Protein Lysate from Prostate Organoids for Western Blot Analysis — TIMING: 2.5-4 H NOTE: Before collecting organoids for protein lysate extraction, prepare and pre-warm dispase-containing media (Table 1). Remove the media from each well by tilting the 24-well plate at a 45° angle and gently removing the existing media from the center of each well using a P1000 pipette while avoidi…

Divulgaciones

The authors have nothing to disclose.

Materials

A83-01  Tocris  2939
Advanced DMEM/F-12  Thermo Fisher Scientific  12634010
B-27 Supplement (50x), Serum Free Thermo Fisher Scientific 17504044
Dispase II, Powder  Thermo Fisher Scientific  17-105-041
Fetal Bovine Serum (FBS)  Sigma  F8667
GlutaMAX Thermo  Fisher Scientific  35050061
Halt Phosphatase Inhibitor  Thermo Fisher Scientific  78428
Matrigel GFR Membrane Matrix Corning CB- 40230C
N-acetyl-L-cysteine  Sigma A9165
Normocin  Thermo Fisher Scientific ant-nr-1
Penicillin-Streptomycin  (10,000 U/
mL)
Thermo Fisher Scientific  15-140-122
Poly(2-hydroxyethyl methacrylate)
(Poly-HEMA)
Sigma  P3932-25G
Radioimmunoprecipitation assay
(RIPA)
Thermo Fisher Scientific  PI89901
Recombinant Human EGF, AnimalFree PeproTech  AF-100-15
Recombinant Human Noggin  PeproTech  120-10C
RPMI 1640 Medium, HEPES (cs of
10)
Thermo Fisher Scientific  22400105
Sonic Dismembrator  Thermo Fisher Scientific FB120
Triton X-100 Sigma X100-5ML
Y-27632 dihydrochloride (ROCK inhibitor)  Selleck Chemical S1049-50MG

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Citar este artículo
Protein Lysate Extraction: A Technique to Lyse Organoids to Collect Cellular Proteins. J. Vis. Exp. (Pending Publication), e20390, doi: (2023).

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