Biomedical Engineering Reference
In-Depth Information
8. Remove the residual fl uids by standing the edge of the slide
glass on a paper towel.
9. Drop 30
L of mounting media onto the slide, and put the
cover slip over the tissue area without making an air bubble.
10. Dry the slide glass in a fl at and dark place for 4-5 h. After com-
pletely dry, clean the surface area of cover glass carefully with
70 % ethanol and Kimwipes.
11. Acquire images by confocal microscopy using the appropriate
excitation and emission settings for each fl uorescent dye. For
fl uorescence quantifi cation, images can be exported as TIFF
fi les and analyzed using image analyzing software (e.g.,
MetaMorph, Molecular Devices Corp.).
ΚΌ
3.4 Introduction
to Cell-Surface
Biotinylation- Based
Assays
Cell-surface expression of the protein of interest can be assessed by
chemical biotin labeling reagents that target specifi c functional
groups or residues, including primary amines, sulfhydryls, carboxyls,
and carbohydrates. Biotinylation reagents consist of biotin, a reac-
tive moiety, and a spacer that cross-links the biotinylation reagent
to the targets. For primary amines, sulfonated N-hydroxysuccinimide
(NHS) ester is used as a reactive moiety. Commonly used spacers
include a cleavable disulfi de (sulfo-NHS-SS-biotin), aminoca-
proic acid (sulfo-NHS-LC-biotin), and polymers of 4 or 12 poly-
ethylene glycol (PEG) moieties (sulfo-NHS-PEO4-biotin or
sulfo-NHS-PEO12-biotin, respectively;
https://www.piercenet.
com/method/biotinylation
)
. The reactive NHS ester forms an
amide bond with exposed lysine side chains (~7 % of protein is
lysine) or the unblocked N-terminus of polypeptides on the cell
surface (
see
Note 2
). The biotinylated proteins are frequently
precipitated on avidin beads for electrophoresis and subsequently
analyzed by immunoblot analysis (
see
Note 3
). The cell-surface
biotinylation technique can be also applied to estimate the internal-
ization or recycling of cell-surface proteins by using biotin-stripping
agents, such as MESNA [
9
] (
see
Note 4
). In addition, information
about carbohydrate groups attached to the surface proteins is help-
ful for identifying the intracellular traffi cking route of the protein.
Therefore, the cell-surface biotinylation technique is frequently
combined with glycosidase treatments to analyze the conventional
and unconventional traffi cking of CFTR and other membrane pro-
teins. Finally, various treatments that affect intracellular protein
traffi cking are used to investigate the basic characteristics of intra-
cellular traffi cking pathways in combination with the cell-surface
biotinylation technique (
see
Note 5
). Some exemplary methods are
introduced in Subheading
3.8
.
1. Wash cells transfected with plasmids for expressing CFTR (or
other proteins of interest) or epithelial cells grown on a perme-
able support twice with PBS-CM. In the case of some epithelial
3.5 Cell-Surface
Biotinylation
