Chemiluminescent Western Blot Assay for Protein Processing

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<…

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