Protein Extraction and Proteolysis: A Method to Obtain Proteins from Prostate Tumor Tissue Samples and their Enzymatic Digestion into Peptides
Published: April 30, 2023
Abstract
Source: Cheng, L. C. et al. Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer. J. Vis. Exp. (2018)
This video describes the technique of protein extraction and digestion to obtain peptides from prostate tumor tissue. These peptides on further analysis can help identify novel targets for oncotherapy.
Protocol
1. Protein Extraction Prepare lysis buffer (Table 1). (The volume depends on the number of samples to be harvested.) Harvesting tissues Weigh the tumor and add 2 mL of ice-cold lysis buffer for every 100 mg of tissue in a culture test tube. (Typically, 50 to 150 mg of tissue wet weight is needed.) Homogenize the lysate using a hand-held or benchtop homogenizer (pulse 2x for 15 s). Clean the homogenizer before the first sample and …
Divulgaciones
The authors have nothing to disclose.
Materials
| Ultra-Low Temperature Freezer | Panasonic | MDF-U76V | |
| Freezer -20 °C | VWR | scpmf-2020 | |
| Swing rotor bucket | ThermoFisher Scientific | 75004377 | |
| End-over-end rotator | ThermoFisher Scientific | 415110Q | |
| Low protein-binding Eppendorf tubes | Eppendorf | 22431081 | |
| Trypsin, TPCK Treated | Worthington Biochemicals | LS003740 | |
| Lysyl Endopeptidase | Wako Pure Chemical Industries, Ltd. | 125-05061 | |
| MilliQ water | deionized water used to prepare all solutions and bufferes | ||
| Sonic Dismembrator | Fisher Scientific | FB-120 | sonicator |
| Polytron System PT | Kinematica AG | PT 10-35 GT | homogenizer |
Citar este artículo
Protein Extraction and Proteolysis: A Method to Obtain Proteins from Prostate Tumor Tissue Samples and their Enzymatic Digestion into Peptides. J. Vis. Exp. (Pending Publication), e20403, doi: (2023).