Chemiluminescent Western Blot Assay for Protein Processing
Published: May 31, 2023
Abstract
Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.
Protocol
T-cell experiments were performed according to the ethical agreement 42502-2-1273 Uni MD. 1. Preparing cells for the experiment NOTE: The average number of cells for this immunoprecipitation is 1 × 107. Adherent cells have to be seeded one day before the experiment so that there are 1 × 107 cells on the day of the experiment. Preparing adherent cells for the experiment Seed 5-8 × 106<…
開示
The authors have nothing to disclose.
Materials
| 12.5% SDS gel | Self-made | For two separating gels: 3.28 mL distilled H2O 2.5 mL Tris; pH 8.8; 1.5 M 4.06 mL acrylamide 100 µL 10% SDS 100 µL 10% APS 7.5 µL TEMED for two collecting gels: 3.1 mL distilled H2O 1.25 mL Tris; pH 6.8; 1.5 M 0.5 mL acrylamide 50 µL 10% SDS 25 µL 10% APS 7.5 µL TEMED | |
| 14.5 cm cell dishes | Greiner | 639160 | |
| Acrylamide | Carl Roth | A124.1 | |
| Aerosol filter pipet tips with high recovery filter and wide orifice | VWR | 46620-664 (North America) or 732-0559 (Europe) | Used for pipetting beads to the lysate |
| Used for pipetting beads to the lysate | Sigma Aldrich | A2103 | Dilution: 1:4000 in PBST + 1:100 NaN3 |
| anti-APO-1 Ab | Provided in these experiments by Prof. P. Krammer or can be purchased by ENZO | ALX-805-038-C100 | Used only for immunoprecipitation |
| anti-caspase-10 Ab | Biozol | MBL-M059-3 | Dilution: 1:1000 in PBST + 1:100 NaN3 |
| anti-caspase-3 Ab | Cell signaling | 9662 S | Dilution: 1:2000 in PBST + 1:100 NaN3 |
| anti-caspase-8 Ab C15 | Provided in these experiments by Prof. P. Krammer or can be purchased by ENZO | ALX-804-242-C100 | Dilution: 1:20 in PBST + 1:100 NaN3 |
| anti-CD95 Ab | Santa Cruz | sc-715 | Dilution: 1:2000 in PBST + 1:100 NaN3 |
| anti-c-FLIP NF6 Ab | Provided in these experiments by Prof. P. Krammer or can be purchased by ENZO | ALX-804-961-0100 | Dilution: 1:10 in PBST + 1:100 NaN3 |
| anti-FADD 1C4 Ab | Provided in these experiments by Prof. P. Krammer or can be purchased by ENZO | ADI-AAM-212-E | Dilution: 1:10 in PBST + 1:100 NaN3 |
| anti-PARP Ab | Cell signaling | 9542 | Dilution: 1:1000 in PBST + 1:100 NaN3 |
| APS | Carl Roth | 9592.3 | |
| β-mercaptoethanol | Carl Roth | 4227.2 | |
| Bradford solution Protein Assay Dye Reagent Concentrate 450ml | Bio Rad | 500-0006 | Used according to manufacturer's instructions |
| CD95L | Provided in these experiments by Prof. P. Krammer or can be purchased by ENZO | ALX-522-020-C005 | |
| Chemiluminescence detector Chem Doc XRS+ | Bio Rad | ||
| Complete Protease Inhibitor Cocktail (PIC) | Sigma Aldrich | ALX-522-020-C005 | Prepared according to manufacturer's instructions |
| DPBS (10x) w/o Ca, Mg | PAN Biotech | P04-53500 | Dilution 1:10 with H2O, storage in the fridge |
| Electrophoresis buffer | Self-made | 10x electrophoresis buffer: 60.6 g Tris 288 g glycine 20 g SDS ad 2 L H2O 1:10 dilution before usage | |
| Glycine | Carl Roth | 3908.3 | |
| Goat Anti-Mouse IgG1 HRP | SouthernBiotech | 1070-05 | Dilution 1:10.000 in PBST + 5% milk |
| Goat Anti-Mouse IgG2b HRP | SouthernBiotech | 1090-05 | Dilution 1:10.000 in PBST + 5% milk |
| Goat Anti-Rabbit IgG-HRP | SouthernBiotech | 4030-05 | Dilution 1:10.000 in PBST + 5% milk |
| Interleukin-2 Human (hIL-2) | Merckgroup/ Roche | 11011456001 | For activation of T cells |
| KCl | Carl Roth | 6781.2 | |
| KH2PO4 | Carl Roth | 3904.1 | |
| Loading buffer 4x Laemmli Sample Buffer, 10 mL | Bio Rad | 161-0747 | Prepared according to manufacturer's instructions |
| Luminata Forte Western HRP substrate | Millipore | WBLUFO500 | |
| Lysis buffer | Self-made | 13.3 mL Tris-HCl; pH 7.4; 1.5 M 27.5 mL NaCl; 5 M 10 mL EDTA; 2 mM 100 mL Triton X-100 add 960 mL H2O | |
| Medium for adherent cells DMEM F12 (1:1) w stable Glutamine, 2,438 g/L | PAN Biotech | P04-41154 | Adding 10% FCS, 1% Penicillin-Streptomycin and 0.0001% Puromycin to the medium |
| Medium for primary T cells | gibco by Life Technologie | 21875034 | Adding 10% FCS and 1% Penicillin-Streptomycin to the medium |
| Milk powder | Carl Roth | T145.4 | |
| Na2HPO4 | Carl Roth | P030.3 | |
| NaCl | Carl Roth | 3957.2 | |
| PBST | Self-made | 20x PBST: 230 g NaCl 8 g KCl 56.8 g Na2HPO4 8 g KH2PO4 Copyright © 2021 JoVE Journal of Visualized Experiments jove.com Page 3 of 3 20 mL Tween-20 ad 2 L H2O dilution 1:20 before usage | |
| PBST + 5% milk | Self-made | 50 g milk powder + 1 L PBST | |
| PHA | Thermo Fisher Scientific | R30852801 | For activation of T Cells |
| Power Pac HC | Bio Rad | ||
| Precision Plus Protein Standard All Blue | Bio Rad | 161-0373 | Use between 3-5 µL |
| Protein A Sepharose CL-4B beads | Novodirect/ Th.Geyer | GE 17-0780-01 | Affinity resin beads prepared according to manufacturer's instructions |
| Scraper | VWR | 734-2602 | |
| SDS | Carl Roth | 4360.2 | |
| Shaker | Heidolph | ||
| Sodium azide | Carl Roth | K305.1 | |
| TEMED | Carl Roth | 2367.3 | |
| Trans Blot Turbo mini-size transfer stacks | Bio Rad | 170-4270 | Used according to manufacturer's instructions |
| TransBlot Turbo 5x Transfer Buffer | Bio Rad | 10026938 | Prepared according to manufacturer's instructions |
| TransBlot Turbo Mini-size nictrocellulose membrane | Bio Rad | 170-4270 | Used according to manufacturer's instructions |
| Trans-Blot-Turbo | Bio Rad | ||
| Tris | Chem Solute | 8,08,51,000 | |
| Triton X-100 | Carl Roth | 3051.4 | |
| Tween-20 | Pan Reac Appli Chem | A4974,1000 |
