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

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).

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 avoiding the matrix gel ring.
2. To collect organoids, repeatedly blast the matrix gel by pipetting 1 mL of dispase-containing media directly onto th....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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