Assessing the Palmitoylation State of Cell Membrane Proteins from Mouse Brain Tissue

Abstract

Source: Speca, D. J., et al. Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins. J. Vis. Exp. (2020).

The video demonstrates the analysis of the palmitoylation state of cell membrane proteins isolated from mouse brain tissue. The process involves exposing the non-palmitoylated cysteines and blocking them, then using a polymer that binds to the palmitoylated cysteines, aiding in assessing the palmitoylation state of the isolated proteins.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.  

1. Preparation of mouse brain membranes

1. Decapitate mouse using a guillotine apparatus and dissect brain out rapidly (in <1 min, if possible, to minimize palmitoylation changes that may occur during dissection procedure). Homogenize immediately in 10 mL of homogenization buffer (Table 1) in a glass homogenizer (~12 strokes) on ice.
2. Centrifuge lysates for 15 min at 1,400 x g at 4 °C. Transfer the supernatant to a new tube on ice. ....

Results

Table 1: Solutions used for APEGS assay. Homogenization buffer and buffer A can be prepared ahead of time; however, add protease inhibitors and phenylmethylsulfonyl fluoride (PMSF) immediately before use. All other solutions should be prepared fresh.

Buffer

Components

Working conc.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hydroxylamine (HAM)	ThermoFisher	26103
Methoxy-PEG-(CH2)3NHCO(CH2)2-MAL (mPEG)	NOF	ME-050MA	MW ~5000 kDa
Microfuge	Eppendorf	5415R	or equivalent equipment
N-ethylmalemide (NEM)	Calbiochem	34115	Highly toxic
Optical imager for densitometry	Azure Biosystems	Sapphire Biomolecular Imager	or equivalent equipment
Polypropylene tubes with cap	Fisher Scientific	14-956-1D
Serological pipets (glass)	Fisher Scientific	13-678-27D
Table top centrifuge	Beckman	Allegra X-15R	or equivalent equipment
Tris(2-carboxyethyl)phosphinehydrochloride (TCEP)	EMD Millipore	580560