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Methods and Protocols, Volume 3, Issue 4

December 2020 - 21 articles

Cover Story: E. coli strains and T7-promoter-based plasmids have been widely used for the expression of recombinant proteins. However, the limited amount of soluble protein fused with core-streptavidin made it challenging for the immobilization of protein on biotinylated materials. In this study, a T7-promoter-based pET-30a(+) plasmid encoded with a chimeric thymidine phosphorylase (TP)-coreSA gene was constructed as a model system for the expression of TP-coreSA fusion protein. Various bacterial strains with the T7 expression system were used to analyze the expression of soluble fusion protein, with the goal of minimizing time- and cost-consuming steps of protein purification. Our results indicate that the pET-30a(+)-TP-coreSA/Lemo21(DE3) system could provide efficient expression of soluble TP-coreSA fusion protein for purification. The eluted fusion protein tethered on biotinylated A549 cancer cells revealed its functionality by effectively killing cells after loading with prodrug 5′-DFUR.
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Articles (21)

  • Review
  • Open Access
7 Citations
4,990 Views
12 Pages

Development of In Vitro Corneal Models: Opportunity for Pharmacological Testing

  • Valentina Citi,
  • Eugenia Piragine,
  • Simone Brogi,
  • Sara Ottino and
  • Vincenzo Calderone

2 November 2020

The human eye is a specialized organ with a complex anatomy and physiology, because it is characterized by different cell types with specific physiological functions. Given the complexity of the eye, ocular tissues are finely organized and orchestrat...

  • Protocol
  • Open Access
4 Citations
3,507 Views
14 Pages

Effects of Implementing the Timed and Targeted Counselling Model on Pregnancy Outcomes and Newborn Survival in Rural Uganda: Protocol for a Quasi-Experimental Study

  • Geoffrey Babughirana,
  • Sanne Gerards,
  • Alex Mokori,
  • Benon Musasizi,
  • Nathan Isabirye,
  • Isaac Charles Baigereza,
  • Grace Rukanda,
  • Emmanuel Bussaja,
  • Stef Kremers and
  • Jessica Gubbels

29 October 2020

Background: Although mortality rates have declined in Uganda over the last decade, maternal mortality is still high at 336 deaths per 100,000 live births, as is infant mortality at 43 deaths per 1000 live births. One in every 19 babies born in Uganda...

  • Protocol
  • Open Access
2 Citations
4,336 Views
11 Pages

28 October 2020

Butterflies are well known for their beautiful wings and have been great systems to understand the ecology, evolution, genetics, and development of patterning and coloration. These color patterns are mosaics on the wing created by the tiling of indiv...

  • Study Protocol
  • Open Access
2 Citations
2,873 Views
8 Pages

Study Protocol of a Randomized Controlled Trial of Home Modification to Prevent Home Fall Injuries in Houses with Māori Occupants

  • Michael Keall,
  • Hope Tupara,
  • Nevil Pierse,
  • Marg Wilkie,
  • Michael Baker,
  • Philippa Howden-Chapman and
  • Chris Cunningham

23 October 2020

Worldwide, injuries due to falls in the home impose a substantial burden and merit considerable effort to find effective prevention measures. The current study is one of very few randomized controlled trials that assess the effectiveness of home modi...

  • Protocol
  • Open Access
6 Citations
3,274 Views
8 Pages

Ultrasonic Navigation for the Treatment of Medication-Related Jaw Osteonecrosis Involving the Inferior Alveolar Nerve: A Case Report and Protocol Review

  • Gerardo Pellegrino,
  • Francesca Pavanelli,
  • Agnese Ferri,
  • Giuseppe Lizio,
  • Roberto Parrulli and
  • Claudio Marchetti

21 October 2020

Dynamic navigation (DN) is a computer-guided technique employed in different surgical fields and recently adopted in dental implantology to improve the accuracy of dental implant insertion. Medication-related osteonecrosis of the jaws (MRONJ) often r...

  • Protocol
  • Open Access
9 Citations
20,355 Views
9 Pages

An Efficient Method for Isolation of Plasmid DNA for Transfection of Mammalian Cell Cultures

  • Daniel V. Kachkin,
  • Julia I. Khorolskaya,
  • Julia S. Ivanova and
  • Aleksandr A. Rubel

14 October 2020

In this article, we present several protocols that describe the steps from cloning and obtaining a large amount of pure plasmid DNA to generation of lentiviruses based on these constructs. The protocols have been worked out on human cell culture HEK2...

  • Protocol
  • Open Access
13 Citations
3,811 Views
9 Pages

KidneyCare Guided Immuno-Optimization in Renal Allografts: The KIRA Protocol

  • Jennifer N. Gray,
  • Theresa Wolf-Doty,
  • Nimisha Sulejmani,
  • Osama Gaber,
  • David Axelrod,
  • Basmah Abdalla and
  • Gabriel Danovitch

30 September 2020

Immunosuppressant agents are essential in every transplant recipient’s care yet walking the fine line of over- or under-immunosuppression is a constant struggle for both patients and transplant providers alike. Optimization and personalization...

  • Protocol
  • Open Access
27 Citations
8,637 Views
12 Pages

Fibrin Clot Formation and Lysis in Plasma

  • Julie Brogaard Larsen and
  • Anne-Mette Hvas

25 September 2020

Disturbance in the balance between fibrin formation and fibrinolysis can lead to either bleeding or thrombosis; however, our current routine coagulation assays are not sensitive to altered fibrinolysis. The clot formation and lysis assay is a dynamic...

  • Protocol
  • Open Access
4 Citations
3,156 Views
9 Pages

Application of a Flow-Induced Stress Wave and Investigation of Associated Injuries on Cell Monolayers Using a Parallel Plate Flow Chamber

  • Samar Shurbaji,
  • Mahmoud Khatib A. A. Al-Ruweidi,
  • Fatma Hassan Ali,
  • Fatiha M. Benslimane and
  • Huseyin C. Yalcin

24 September 2020

Parallel plate flow chambers are widely used to expose cultured cells to physiological flows for the investigation of a variety of diseases. These applications usually involve the generation of continuous and steady fluid flow over cell monolayers fo...

  • Protocol
  • Open Access
35 Citations
5,821 Views
14 Pages

24 September 2020

Oxidative stress is prominent in many neurodegenerative diseases. Along with mitochondrial dysfunction and pathological protein aggregation, increased levels of reactive oxygen and nitrogen species, together with impaired antioxidant defense mechanis...

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Methods Protoc. - ISSN 2409-9279