Revising Basic Laboratory Methods in a Regulated Environment
I recently finished updating one of InnovATEBIO’s Courses-in-a-Box, Basic Laboratory Methods in a Regulated Environment. This course introduces students to fundamental laboratory methods that preprofessional biotechnologists must master. Over the years, my colleagues and I have tried to identify the most basic concepts and skills that our students require to be successful in various biotech workplaces. One of the first skills that we identified is the ability to prepare aqueous solutions. The box below is an interesting example of why the proper preparation of aqueous solutions should never be taken for granted. As summarized in the box, Ignatoski and Verderame demonstrated that sometimes subtle changes in the composition of their lysis buffers (ionic strength, pH, and component concentrations) profoundly impacted their research conclusions. As instructors, we know there are a number of potential pitfalls in solution preparation. We have found that it takes time and attention for students to learn to prepare biological solutions properly and consistently. Therefore, early in the development of our Basic Laboratory Methods course, we put in a unit on solution preparation. But wait, what does a technologist need to know before beginning to prepare solutions? It turns out, many things, including:
- How to perform calculations relating to concentration, including calculations involving solutions with more than one component (a task not taught in students’ chemistry courses).
- How to interpret biological solution “recipes” that are often written in a shorthand format.
- How to weigh out chemicals accurately and consistently, including understanding operation, maintenance, and calibration of balances and the impact of factors such as temperature and static charge.
- How to measure out volumes accurately and consistently.
- How to prepare/obtain ultrapurified water
- How to bring a buffer to the proper pH accurately and consistently.
- Etc.
And so our Basic Laboratory Methods in a Regulated Environment course evolved. It now consists of an introduction to metrology, solution preparation, spectrophotometry, and principles and execution of spectrophotometric assays. We also introduce basic separation methods in this course, centrifugation, and filtration. This course serves as the foundation for further courses in molecular biology, cell culture, protein methods, chromatography, and bioprocessing.
The reason we call this course Basic Laboratory Methods in a Regulated Environment is because principles of quality are woven throughout. Students develop a quality “mind-set” and learn to work thoughtfully with awareness of the many factors that can cause error and inconsistency. This approach helps prepare students to work in a company that is compliant with quality systems, including the Food and Drug Administration’s regulations. The same principles apply to all biotechnology settings – after all, no one wants a product that is not of good quality. Research laboratories produce a product—knowledge—which must be of good quality to be useful. In recent years, the scientific community has become aware of, and in some cases alarmed by, the irreproducibility of some critically important published studies. In October 2022, the National Science Foundation responded to this concern in a Dear Colleague letter stating: “… NSF reaffirms its commitment to advancing reproducibility and replicability in science…” Quality, reproducibility, and replicability are inextricably linked in the regulated biopharmaceutical realm and in the research world. Our students, regardless of their ultimate career destination, need to begin on their pathway with a thoughtful approach to the fundamentals.
The Importance of Lysis Buffer Components
A study that investigated the effects of buffer components was reported by Ignatoski and Verderame. (Ignatoski, Kathleen M. Woods, and Michael F. Verderame. “Lysis Buffer Composition Dramatically Affects Extraction of Phosphotyrosine-Containing Proteins.” BioTechniques, vol. 20, no. 5, 1996, pp. 794–96. Crossref, doi:10.2144/96205bm13.) The ultimate goal of these researchers was to compare proteins in cultured cells that were transformed with an oncogene to proteins in cultured cells that were not transformed. The work required lysing the cells with lysis buffer to release their proteins into solution. The scientists discovered that the components of the lysis buffer had a significant effect on which proteins they detected and therefore, their conclusions regarding differences between transformed and non-transformed cells.
Ignatoski and Verderame compared the effects of three different lysis buffers. All three buffers included protease inhibitors and also the following ingredients:
| Lysis Buffer A | Lysis Buffer B | Lysis Buffer C |
| 50 mM Tris-HCl, pH 7.5 | 10 mM Tris-HCl, pH 7.4 | 30 mM Tris-HCl pH 6.8 |
| 150 mM NaCl | 50 mM NaCl | 150 mM NaCl |
| 1% Nonidet P-40 (detergent) | 50 mM NaF | 1% NP40 (detergent) |
| 0.25% Na+ deoxycholate (detergent) | 1% Triton X-100 (detergent) | 0.5% Na+ deoxycholate (detergent) |
| 10 mg/mL BSA | 5 mM EDTA | 0.1% SDS (detergent) |
| 150 mM Na3VO4 (inhibits phosphatase enzymes) | ||
| 30 mM Na4P2O7 |
The authors observed that the three lysis buffers appear to “be solubilizing different subsets of cellular proteins” and they postulated that:
1. The type and concentration of detergent could have an effect on which proteins are detected.
2. The ionic strength of the buffer plays a critical role in protein solubilization.
3. pH can affect detergent solubility, especially of Na+ deoxycholate, thereby changing which proteins are solubilized.
4. The buffer effectiveness may have been affected by the presence of phosphate.
The authors concluded that the lysis buffer components can play a role in affecting experimental results and therefore “several different lysis buffers . . . should be tested to obtain optimal experimental conditions.” It is clear from this example that experimental reproducibility requires careful attention to the solutions that are used. If various researchers use subtly different lysis buffers, they might reach dramatically different conclusions about the proteins involved in cancer.