eoe sub articles

EoE Sub Articles

1. Functionalization of the array with the biotinylated antibody
<ol>
	<li>Prepare the following solutions.
	<ol>
		<li>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.</li>
		<li>T50-BSA by supplementing T50 buffer with 0.1 mg/mL of BSA. Keep the prepared solution on ice.</li>
		<li>10 mg/mL of sodium borohydride (NaBH4) in 1x PBS. Prepare this immediately before use.</li>
		<li>0.2 mg/mL of NeutrAvidin (see Table of Materials) in T...

single-molecule pull-down assay for protein phosphorylation analysis: a high throughput technique to quantify protein phosphorylation in cell lysate

Source: Bailey, E. M. et al., <a href="https://www.jove.com/v/63665">An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation.</a> 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.

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

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

The phosphorylation of certain cellular proteins is important for their biological function.
To quantify the phosphorylation levels of intact proteins using a single-molecule pull-down assay, begin by taking a glass coverslip with a grid-array pattern. Each compartment is coated with biotinylated polyethylene glycol or PEG-based linkers interspaced with normal PEG to prevent steric hindrance during protein binding.
Treat the coverslip with a reducing reagent to minimize the autofluorescence in analysis. Overlay the coverslip with Neutravidins - biotin-binding proteins - and incubate, allowing them to bind biotinylated PEG linkers.
Supplement the array with specific anti-target protein antibodies. Each antibody contains biotin linked to its Fc region, which helps anchor the antibody to the neutravidin, causing immobilization.
Now, add the cell culture lysate containing various proteins, including the green fluorescence-tagged phosphorylated and non-phosphorylated variants of the target protein, and incubate. Both variants of the target protein get captured by the antibodies, forming complexes. Wash to remove the unbound proteins.
Next, treat these complexes with the second set of red fluorescence-tagged antibodies, which exclusively bind the phosphorylated residues of the protein, imparting a different fluorescence signal to the complex.
Image the array under a fluorescence microscope and observe for two distinct fluorescence signals. The colocalization of both fluorescence signals indicates the presence of phosphorylated proteins in the sample.

single-molecule-pull-down-assay-for-protein-phosphorylation-analysis

Research

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Biological Techniques

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Immunoassays

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

JoVE Journal

Biochemistry

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...

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

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