A STAGE Tip Procedure for Desalting and Purification of Peptides
Published: February 29, 2024
Abstract
This video demonstrates peptide purification through the STAGE Tip procedure. A miniature column containing resin beads bonded to long hydrocarbon chains facilitates hydrophobic interaction-based purification. Upon loading a pre-digested peptide sample into the column, the peptide fragments bind to the resin beads, allowing impurities to pass through. An elution buffer that disrupts the hydrophobic interaction is applied to elute the peptide fragments.
Protocol
1. Purification and Desalting of Peptides Pack three layers of C18 resin in a 200 μl pipette tip for each peptide sample to construct a STop And Go Extraction (STAGE)-tip. Add 1/10 stopping solution (20% acetonitrile [ACN)] and 6% trifluoroacetic acid [TFA]) to samples. Centrifuge samples for pellet precipitation at 13,200 rpm for 10 min. Transfer the supernatant to a new 2 mL tube and discard the pellet. Wash stage tips with 100 μl of 100% AC…
Açıklamalar
The authors have nothing to disclose.
Materials
| 15 ml Falcon Tube | VWR | 470225-000 | |
| 25 mL Serological | Fisher Scientific | 14955235 | |
| Ammonium Bicarbonate (ABC) | ThermoFisher Scientific | A643-500 | |
| Acetic Acid | Sigma Aldrich | 1099510001 | |
| Acetone | Sigma Aldrich | 34850-1L | |
| Acetonitrile (ACN), MS grade | Pierce | TS-51101 | |
| BSA assay kit | VWR | CA62408-940 | |
| C18 disks (STAGE tips) | Fisher Scientific | 13110019 | |
| Centrifuge 5810 R | Sigma Aldrich | EP022628188 | |
| Dithiothreitol (DTT) | ThermoFisher Scientific | R0861 | Prepare 1M stock of DTT aliquots and store at -20°C |
| Fetal Bovine Serum (FBS) | ThermoFisher Scientific | 12483020 | Thaw out, prepare aliquots and stored at -20°C |
| HEPES | Sigma Aldrich | H3375 | Prepare 8M Urea/40m HEPES aliquots, store at -20 °C |
| High-resolution mass spectrometer | ThermoFisher Scientific | 726042 | |
| Iodoacetamide (IAA) | Fisher Scientific | AAA1471514 | Prepare 0.55M stock of IAA aliquots and store at -20°C. Avoid light exposure |
| L-glutamine | Fisher Scientific | 25030081 | Thaw out, prepare aliquots and stored at -20°C |
| LoBind Microcentrifuge tubes | Eppendorf | 13-698-794 | |
| MaxQuant | https://www.maxquant.org/ | ||
| PCR strip tubes | VWR | 76318-802 | |
| Penicillin Streptomycin, Amphotericin | Sigma Aldrich | A5955 | Thaw out, prepare aliquots and stored at -20°C |
| Perseus | https://www.maxquant.org/Perseus/ | ||
| phosphate-buffered solution (PBS) | Bioshop | PBS408 | Store at room temperature |
| Probe sonicator | Fisher Scientific | 100-132-894 | |
| Protease inhibitor cocktail (PIC) tablet | Sigma Aldrich | 5892791001 | Store at 4°C |
| RPMI 1640 | ThermoFisher Scientific | 11875093 | Store at 4°C |
| Sodium Dodecyl Sulphate (SDS) | ThermoFisher Scientific | J18220.A1 | prepare 2% SDS solution |
| SpeedVac Centrifuge | Eppendorf | 07-748-15 | |
| STAGE tipping centrifuge | Sonation | STC-V2 | |
| T75 Flasks | ThermoFisher Scientific | 156499 | |
| Trifluoroacetic acid | ThermoFisher Scientific | 85183 | |
| Tris HCL pH 8.5 | Fisher Scientific | BP152-1 | Store at 4°C |
| Trypsin/LysC | Thermo Fisher Scientific | A40009 | aliquot and store at -20°C |
| Ultrasonic bath | VWR | 97043-960 | |
| Urea | Bioshop | URE001.1 |
Etiketler
Bu Makaleden Alıntı Yapın
A STAGE Tip Procedure for Desalting and Purification of Peptides. J. Vis. Exp. (Pending Publication), e22210, doi: (2024).