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