Protocol for detecting LDLR on the cell surface of primary mouse hepatocytes using cell-surface biotinylation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Plasma membrane proteins play crucial roles in cellular functions.
• Studying these proteins is essential for understanding cellular processes and disease mechanisms.
• Current methods for isolating plasma membrane proteins can be complex and inefficient.

Purpose of the Study:

• To develop a protocol for selective biotinylation, solubilization, and enrichment of plasma membrane proteins from primary mouse hepatocytes.
• To enable the study of specific cell surface proteins, such as the low-density lipoprotein receptor (LDLR).

Main Methods:

• Adeno-associated virus subtype 8 (AAV8) injection in C57/BL6 mice for hepatocyte-specific gene knockdown.
• Surface protein labeling using EZ-LINK Sulfo-NHS-LC-Biotin.
• Immunoprecipitation for enrichment of target proteins like LDLR.
• Streptavidin-HRP based detection for assessing surface LDLR levels.

Main Results:

• The protocol allows for selective isolation and enrichment of plasma membrane proteins.
• Successful knockdown of target genes in hepatocytes was achieved using AAV8.
• Quantification of surface LDLR was demonstrated using streptavidin-HRP.

Conclusions:

• This protocol provides a robust method for studying plasma membrane proteins in hepatocytes.
• The technique facilitates the investigation of protein function and interactions in a cellular context.
• This method can be adapted for studying various cell surface proteins and their roles in physiological and pathological conditions.