The Penn State Protein Ladder system for inexpensive protein molecular weight markers | JoVE Visualize

The Penn State Protein Ladder system for inexpensive protein molecular weight markers

Ryan T Santilli^1,2, John E Williamson¹, Yoshitaka Shibata^1,2

¹Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802, USA.

Scientific Reports|August 19, 2021

View abstract on PubMed

Summary

Researchers developed the Penn State Protein Ladder system for cost-effective protein molecular weight markers. This system uses engineered E. coli plasmids to produce high-yield protein standards for SDS-PAGE and Western blots.

Area of Science:

Biochemistry
Molecular Biology
Protein Expression Systems

Background:

Accurate protein molecular weight determination is crucial for biochemical research.
Existing protein ladder systems can be expensive and difficult to produce.
Need for accessible and affordable protein standards for various laboratory applications.

Purpose of the Study:

To develop an economical and user-friendly system for producing protein molecular weight markers.
To create a versatile tool for recombinant protein expression and purification.
To provide reliable reagents for both research and teaching laboratories.

Main Methods:

Engineered plasmids for expressing a range of protein molecular weight markers (10-250 kD) in E. coli.
Utilized histidine tags for straightforward protein purification.
Incorporated engineered immunoglobulin binding domains for direct detection on Western blots.
Developed polycistronic expression vectors for coexpressing multiple protein markers.

Main Results:

Successfully produced protein markers from 10 kD to 250 kD with high expression levels (10-50 mg/L).
Demonstrated appropriate migration of all expressed proteins on SDS-PAGE gels.
Achieved efficient purification using histidine tags and direct detection via Western blots.
Quantified high-throughput production capacity, with 50 ml culture yielding thousands of lanes.

Conclusions:

The Penn State Protein Ladder system offers an inexpensive and efficient method for generating protein molecular weight markers.
The system provides versatile reagents suitable for recombinant protein expression, purification, and quality control.
These expression plasmids serve as valuable tools for educational purposes and research laboratories.