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

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記事を引用
Chemiluminescent Western Blot Assay for Protein Processing. J. Vis. Exp. (Pending Publication), e21369, doi: (2023).

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