Peptide Purification: An RP-HPLC-based Technique to Extract Peptides from Digested Protein Lysates
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)
In this video, we demonstrate reversed-phase high-performance liquid chromatography to purify peptides from a digested protein lysate mixture. These purified peptides are then lyophilized and stored for downstream applications. Analysis of peptides can help understand the mechanics involved in cellular signaling and cancer.
Protocol
1. Reverse Phase Extraction Start with a pre-digested protein lysate in a glass vial. Filter the sample by using a 15 mL 10 kDa cutoff filter. Centrifuge the sample at 3,500 x g using the swing bucket rotor (or 3,500 x g in a fixed angle rotor) at 15 °C until the retentate volume is less than 250 µL (this takes approximately 45 – 60 min). Collect the flow-through and discard the retentate. NOTE: The experiment can be paused …
Açıklamalar
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 | |
| Vacuum manifold | Restek | 26080 | |
| Lyophilizer | Labconco | 7420020 | |
| CentriVap Benchtop Vacuum Concentrator | Labconco | 7810010 | |
| Amicon Ultra-15 Centrifugal Filter Units | Millipore Sigma | UFC901024 | |
| End-over-end rotator | ThermoFisher Scientific | 415110Q | |
| Glass culture tubes | Fisher Scientific | 14-961-26 | |
| Parafilm | Fisher Scientific | 13-374-12 | |
| 20G needle | BD | B305175 | |
| Kimwipes | Fisher Scientific | 06-666A | |
| Screw cap cryotube | ThermoFisher Scientific | 379189 | |
| Nunc 15 mL conical tubes | ThermoFisher Scientific | 12-565-268 | |
| Low protein-binding Eppendorf tubes | Eppendorf | 22431081 | |
| 3 mL syringe | BD | 309657 | |
| Trifluoracetic Acid (TFA) | Fisher Scientific | PI-28904 | |
| Acetonitrile (ACN) | Fisher Scientific | A21-1 | |
| MilliQ water | Deionized water used to prepare all solutions and bufferes |
Bu Makaleden Alıntı Yapın
Peptide Purification: An RP-HPLC-based Technique to Extract Peptides from Digested Protein Lysates. J. Vis. Exp. (Pending Publication), e20404, doi: (2023).