Single-Molecule Pull-Down Assay for Protein Phosphorylation Analysis: A High Throughput Technique to Quantify Protein Phosphorylation in Cell Lysate

Abstract

Source: Bailey, E. M. et al., An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation. J. Vis. Exp. (2022)

This video demonstrates a sensitive quantification technique of protein phosphorylation using a single-molecule pull-down assay. The functionalization of polyethylene glycol-biotin and the use of labeled antibodies increases the detection of phosphorylated tyrosine with specificity.

Protocol

1. Functionalization of the array with the biotinylated antibody

1. Prepare the following solutions.
   1. T50 Buffer, a solution of 10 mM Tris-HCl (pH 8.0) and 50 mM NaCl. The solution is stable for 1 month at RT.
   2. T50-BSA by supplementing T50 buffer with 0.1 mg/mL of BSA. Keep the prepared solution on ice.
   3. 10 mg/mL of sodium borohydride (NaBH₄) in 1x PBS. Prepare this immediately before use.
   4. 0.2 mg/mL of NeutrAvidin (see Table of Materials) in T50 buffer.
      CAUTION: NaBH₄ is a reducing agent and is flammable. Always purge the container with nitrogen gas after....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tubes	MTC Bio	C2000
10 mM Tris-HCl pH 7.4
10 mM Tris-HCl pH 8.0/ 50 mM NaCl			T50 Buffer
100 mm Tissue Culture dish	CELLTREAT	229620	Storage of piranha etched glass/arrays
15 mL conical tube
16% Paraformaldehyde Aqueous Solution	Electron Microscopy Sciences	15710	Hazardous
50 mL conical tube			Functionalized Glass storage/ KOH reuse
8% Glutaraldehyde Aqueous Solution	Electron Microscopy Sciences	16020	Hazardous
Alexa Fluor 647 NHS Ester	Thermo Fisher Scientific	A-20006
Anti-Human EGFR (External Domain) – Biotin	Leinco Technologies, Inc	E101
Anti-p-Tyr Antibody (PY99) Alexa Fluor 647	Santa Cruz Biotechnology	sc-7020 AF647
Biotin-PEG	Laysan Bio	Biotin-PEG-SVA, MW 5,000
Bovine serum albumin	Gold Biotechnology	A-420-1	Tyrode's Buffer Component
Coverslips 24 x 60 #1.5	Electron Microscopy Sciences	63793
emCCD camera	Andor iXon
Halt Phosphotase and Protease Inhibitor Cocktail (100X)	Thermo Fisher Scientific	78446	Lysis Buffer Component
Ice
Immersol 518F immersion oil	Zeiss	444960-0000-000
In-house vacuum line
Mix-n-Stain CF Dye Antibody Labeling Kits	Biotium	92245
Nanogrid	Miraloma Tech
NeutrAvidin Biotin Binding Protein	Thermo Fisher Scientific	31000
Nitrogen (compressed gas)
Olympus iX71 Microscope	Olympus
Parafilm M Sealing Film	The Lab Depot	HS234526C
PBS pH 7.4	Caisson Labs	PBL06
Phospho-EGF Receptor (Tyr1068) (1H12) Mouse mAb	Cell Signaling Technology	2236BF
Quad-view Image Splitter	Photometrics	Model QV2
Semrock emission filters: blue (445/45 nm), green (525/45 nm), red (600/37 nm), far-red (685/40 nm)	Semrock	LF405/488/561/635-4X4M-B-000
Sodium Borohydride (NaBH4)	Millipore Sigma	452874	Hazardous