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

Samenvatting

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 the matrix gel ring until the entire ring is dislodged, and transfer to a 1.5 mL microcentrifuge tube. 
   NOTE: It is critical to avoid direct contact with the Poly-HEMA-coated wells. Direct contact may cause contamination of the collected material with Poly-HEMA, which could negatively impact cell survival.
3. Place the 1.5 mL microcentrifuge tube(s) into a 37 °C 5% CO₂ incubator for 30 min to 1 h to allow complete digestion of the matrix gel by dispase.
4. Pellet down the organoids by centrifugation at 800 x g for 5 min at RT and remove the supernatant using a micropipette.
5. Add phosphate-buffered saline (PBS) to the organoid pellet and resuspend by gently flicking. 
   NOTE: Failure to sufficiently resuspend the organoid pellet may result in the contamination of organoid material with residual dispase or matrix gel.
6. Pellet the organoids by centrifugation at 800 x g for 5 min at RT and remove the supernatant using a micropipette.
7. Fast freeze the organoid pellets by placing each tube into a solution containing dry ice and methanol. Store the tube(s) until future use at -80 °C. Alternatively, extract protein lysate immediately following step 1.6.
8. Resuspend the organoid pellets in 100 µL of protein lysis buffer (Table 1) per 10 µL of packed cell volume. Flick to resuspend. 
   NOTE: If resuming after fast-freezing, ensure protein lysis buffer is thawed before removing samples from -80 °C, as lysis buffer must be added to samples immediately to prevent phosphatase and protease activity.
9. Incubate the samples in protein lysis buffer on ice for at least 45 min. 
   NOTE: It is recommended to sonicate before incubation on ice to increase the efficiency of nuclear protein recovery; however, sonication is not required. If sonication is not performed, proceed to step 1.10.
   1. To sonicate, submerge tubes in wet ice and gently apply the tip of the sonic dismembrator to the microcentrifuge tube. Sonicate for 40 s at 20 kHz.
10. Proceed to Western blot following established protocols.

Table 1: Instructions for the preparation of key solutions

Recipes
Dispase-containing media	1 mg/mL dispase + 10 µM ROCK inhibitor in advanced DMEM F12. Filter sterilize using 0.22 µm filter.
Dissociation media	10% FBS + 1x Penicillin-Streptomycin in RPMI 1640. Filter sterilize using 0.22 µm filter.
Protein lysis buffer	RIPA buffer + phosphatase inhibitors + protease inhibitors
Blocking solution	10% FBS in PBS with 0.2% Triton X-100

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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