Presentation Title

Antimicrobial Peptide Detection Using Acid Urea Polyacrylamide Gel Electrophoresis and Western Immunoblotting

Faculty Mentor

Edith Porter

Start Date

17-11-2018 8:30 AM

End Date

17-11-2018 10:30 AM

Location

CREVELING 27

Session

POSTER 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Jeffrey Knott and Edith Porter

California State University Los Angeles

Department of Biological Sciences

Mentor: Dr. Edith Porter

10/1/2018

Antimicrobial Peptide Detection Using Acid Urea Polyacrylamide Gel Electrophoresis and Western Immunoblotting

Antimicrobial peptides (AMPs) are small cationic effector proteins which are ubiquitously present in the skin and mucosal surfaces. They may be constitutively expressed or upregulated by several orders of magnitude via microbial pattern recognition pathways, cytokine stimulation, and vitamin D. Alveolar epithelial cells are able to provide protective barrier function and our laboratory is focused on identifying novel approaches to augment their antimicrobial function. These mucosal cells express two types of AMPs, known as human beta defensins (HBD1-3) and cathelicidins (LL37) which range in molecular weight between 3.9 kDa and 5.2 kDa. Because some peptides differ by as little as a few hundred Daltons, analysis of alveolar cell excretions are not suitable by conventional SDS gel electrophoresis.

We have set up a detection system using acid urea polyacrylamide gel electrophoresis (AU-PAGE) followed by western immunoblot that allows us to separate these small peptides based on their charge and shape in addition to size. In AU-PAGE, peptides are immersed in an acidic environment in which their cationic properties enable different migration patterns. This enhanced separation is then combined with specific antibody directed detection using western immunoblotting, in which we have been successful differentiating HBD1-3 to the low nanogram range. We are now beginning to analyze secretions of A549 type II alveolar cells stimulated with various cytokines in order to explore how we can modulate AMP productions at this important mucosal interface.

Acknowledgements: Dr. Wuyuan Lu, University of Maryland, for synthetic antimicrobial peptides. NSS Research and Scholarship Award 2018 from the College of Natural and Social Sciences at California State University Los Angeles.

Summary of research results to be presented

Results will include successful detection of HBD1, HBD2 and HBD3 using the methods described in abstract. Human beta defensins obtained from A549 Alveolar cell secretions are being analyzed currently and may be available by conference date, but at this time of writing we have only proof of detection method.

This document is currently not available here.

Share

COinS
 
Nov 17th, 8:30 AM Nov 17th, 10:30 AM

Antimicrobial Peptide Detection Using Acid Urea Polyacrylamide Gel Electrophoresis and Western Immunoblotting

CREVELING 27

Jeffrey Knott and Edith Porter

California State University Los Angeles

Department of Biological Sciences

Mentor: Dr. Edith Porter

10/1/2018

Antimicrobial Peptide Detection Using Acid Urea Polyacrylamide Gel Electrophoresis and Western Immunoblotting

Antimicrobial peptides (AMPs) are small cationic effector proteins which are ubiquitously present in the skin and mucosal surfaces. They may be constitutively expressed or upregulated by several orders of magnitude via microbial pattern recognition pathways, cytokine stimulation, and vitamin D. Alveolar epithelial cells are able to provide protective barrier function and our laboratory is focused on identifying novel approaches to augment their antimicrobial function. These mucosal cells express two types of AMPs, known as human beta defensins (HBD1-3) and cathelicidins (LL37) which range in molecular weight between 3.9 kDa and 5.2 kDa. Because some peptides differ by as little as a few hundred Daltons, analysis of alveolar cell excretions are not suitable by conventional SDS gel electrophoresis.

We have set up a detection system using acid urea polyacrylamide gel electrophoresis (AU-PAGE) followed by western immunoblot that allows us to separate these small peptides based on their charge and shape in addition to size. In AU-PAGE, peptides are immersed in an acidic environment in which their cationic properties enable different migration patterns. This enhanced separation is then combined with specific antibody directed detection using western immunoblotting, in which we have been successful differentiating HBD1-3 to the low nanogram range. We are now beginning to analyze secretions of A549 type II alveolar cells stimulated with various cytokines in order to explore how we can modulate AMP productions at this important mucosal interface.

Acknowledgements: Dr. Wuyuan Lu, University of Maryland, for synthetic antimicrobial peptides. NSS Research and Scholarship Award 2018 from the College of Natural and Social Sciences at California State University Los Angeles.