Bacteriophages pp 107-115 | Cite as

Peptidoglycan Hydrolytic Activity of Bacteriophage Lytic Proteins in Zymogram Analysis

  • Lorena Rodríguez-Rubio
  • David M. Donovan
  • Beatriz Martínez
  • Ana Rodríguez
  • Pilar García
Part of the Methods in Molecular Biology book series (MIMB, volume 1898)


Zymogram or zymography is an electrophoretic technique based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which enables visualization of enzymatically active protein species separated by molecular mass. The strategy is to perform SDS-PAGE on the proteins in question while including an opaque substrate of the enzyme embedded within the polyacrylamide gel. Here, we describe a zymogram protocol for phage lytic proteins (peptidoglycan hydrolases) using peptidoglycan (or whole cells) from a susceptible gram-positive bacterial species as substrate. Proteins are prepared and analyzed simultaneously on two separate gels: First, standard denaturing SDS-PAGE followed by conventional protein staining (e.g., Coomassie) is run to identify the migration pattern of the protein species in the sample; second, the zymogram gel in which either cells or peptidoglycan from a susceptible bacterium have embedded in the SDS gel matrix is performed. After electrophoresis, the SDS is removed from the zymogram gel, allowing the proteins (now separated by molecular mass) to assume an active conformation and ultimately digest the opaque substrate (yielding a nonopaque product). This results in a cleared spot in an otherwise opaque gel which corresponds to the location of an enzymatically active protein species. This assay can be used to qualitatively assay the enzymatic activity of endolysins from cell extracts, or to identify virion-associated peptidoglycan hydrolases in phage particles.

Key words

Enzyme Phage lytic proteins SDS-PAGE Hydrolytic activity 



This research work was supported by grants AGL2012-40194-C02-01 (Ministry of Science and Innovation, Spain), GRUPIN14-139 (Program of Science, Technology and Innovation 2013-2017, Principado de Asturias, Spain), and IS-4573-12 R from the Binational Agricultural Research and Development Fund (BARD). The US Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual’s income is derived from any public assistance program. The USDA is an equal opportunity provider and employer.


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Lorena Rodríguez-Rubio
    • 1
    • 2
  • David M. Donovan
    • 3
  • Beatriz Martínez
    • 1
  • Ana Rodríguez
    • 1
  • Pilar García
    • 1
  1. 1.DairySafe Group, Department of Technology and Biotechnology of Dairy ProductsInstituto de Productos Lácteos de Asturias (IPLA-CSIC)AsturiasSpain
  2. 2.Department of Genetics, Microbiology and StatisticsUniversity of BarcelonaBarcelonaSpain
  3. 3.Animal Biosciences and Biotechnology LaboratoryBeltsville Area Research Center, ARS, NEA, USDABeltsvilleUSA

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