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

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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).

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