Isolation and Digestion of Neutrophil Proteins
Published: February 29, 2024
Abstract
This video demonstrates the protein isolation from neutrophils, which is further processed and digested into smaller peptides. These peptides can be used for future analysis.
Protocol
1. Neutrophil Cell Collection and Lysis Collect neutrophil-differentiated HL60 cells in a 15 mL tube by centrifuging samples at 400 x g for 5 mins. NOTE: Neutrophils should be at a confluency of 3.5 x 105 cells/mL after differentiation. Harvest approximately five million cells for total proteome extraction. Discard the supernatant, and add 15 mL of phosphate-buffered solution (PBS) into the tube. Centrifug…
Representative Results
Figure 1. Workflow of HL60 cells differentiated into neutrophils for mass spectrometry-based proteomics profiling. Undifferentiated and differentiated (via DMSO chemical stimulation) HL60 cell lines were cultured, collected, and processed for total proteome extraction. Proteins were digested with Trypsin/LysC, and peptides were purified using C18 STAGE-tips, followed by detection by LC-MS/MS. Max…
Declarações
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 | |
| Acetone | Sigma Aldrich | 34850-1L | |
| BSA assay kit | VWR | CA62408-940 | |
| 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 |
| Heating block | |||
| HEPES | Sigma Aldrich | H3375 | Prepare 8M Urea/40m HEPES aliquots, store at -20 °C |
| 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 |
| Liquid nitrogen | |||
| LoBind Microcentrifuge tubes | Eppendorf | 13-698-794 | |
| Penicillin Streptomycin, Amphotericin | Sigma Aldrich | A5955 | Thaw out, prepare aliquots and stored at -20°C |
| Neutrophil-differentiated HL60 cells | |||
| 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 | |
| T75 Flasks | ThermoFisher Scientific | 156499 | |
| Tris HCL pH 8.5 | Fisher Scientific | BP152-1 | Store at 4°C |
| Ultrasonic bath | VWR | 97043-960 | |
| Urea | Bioshop | URE001.1 |
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Citar este artigo
Isolation and Digestion of Neutrophil Proteins. J. Vis. Exp. (Pending Publication), e22209, doi: (2024).