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Steric hindrance in immunolabelling.

W F Voorhout, J J Leunissen-Bijvelt, J L Leunissen

Journal of Microscopy

|March 1, 1986

Summary

Immunocytochemical detection of PhoE pore protein in E. coli was hindered by lipopolysaccharide (LPS) in intact cells. Cryosections revealed LPS steric hindrance, emphasizing varied labeling method comparisons for accurate bacterial outer membrane protein detection.

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Area of Science:

Microbiology
Immunocytochemistry
Structural Biology

Background:

The PhoE pore protein is a key component of the outer membrane in Escherichia coli K-12.
Accurate detection of outer membrane proteins is crucial for understanding bacterial cell biology.

Purpose of the Study:

To investigate the accessibility of PhoE pore protein antigenic sites in E. coli K-12.
To evaluate the impact of different immunocytochemical labeling approaches on PhoE detection.
To elucidate the role of lipopolysaccharide (LPS) in steric hindrance of protein detection.

Main Methods:

Immunocytochemistry using various labeling techniques (immuno-gold on cryosections, immunofluorescence, whole-mount, freeze-etch).
Cryo-ultramicrotomy for detailed ultrastructural analysis.
Comparative analysis using wild-type E. coli and an E. coli Gal-U mutant (defective in LPS chain length).

Main Results:

Immuno-gold labeling on cryosections showed uniform PhoE labeling in the outer membrane.
Immunofluorescence, whole-mount, and freeze-etch methods labeled less than 5% of wild-type cells.
Cryosection reincubation and use of an LPS-defective mutant resulted in 100% PhoE labeling across all methods.
Steric hindrance by LPS carbohydrate chains was identified as the cause of limited accessibility in intact cells.

Conclusions:

The lipopolysaccharide (LPS) carbohydrate chains sterically hinder the accessibility of PhoE pore protein antigenic sites in intact wild-type E. coli.
Cryosectioning partially overcomes this steric hindrance by exposing antigenic determinants.
Comparing results from multiple, distinct immunocytochemical labeling approaches is essential for accurate interpretation of outer membrane protein accessibility.

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