Appendix B: Minimum Details to Report for Routine Laboratory Methods

The following are the critical details required to replicate common biochemical techniques. At minimum you must include these details in your methods and captions.

  • Agarose gel electrophoresis
    • Captions: percent agarose; staining methodology; identity of lanes; identity of important bands in reference ladder.
    • Methods: percent agarose; composition and pH of buffer used to make and run the gel; amount of sample loaded; type of reference ladder used; manufacturer of reference ladder; voltage per cm; time; staining procedure; visualization method (i.e. trans-illumination, epi-illumination ), wavelength of light.
  • Chromatography
    • Captions: method (e.g. column, thin layer); stationary phase (e.g. silica, Dowex 50WX4); composition of mobile phase; visualization procedure; identity of samples.
    • Methods: method (e.g. column, thin layer); identity (e.g. silica, Dowex 50WX4) and amount (e.g. height and diameter for column, dimensions for TLC plate) of stationary phase; composition of mobile phase; flow rate and gradients (for column); total time (for plate); visualization procedure.
  • Compositions of solutions
    • Methods: state composition solutions and buffers (e.g. 100 mM Tris buffer, pH 8.50 with 50 mM NaCl); you do not need to describe the recipes to make concentrated stocks and steps you carried-out to prepare the buffer.
  • Data plot (general)
    • Captions: if a figure has a trendline, give the equation of the trendline and the R2 value in the caption.
    • Methods: describe your methods of data analysis, important equations, and calculations used. Define non-standard units. If you analyzed and plotted your data using spreadsheet software, you must name the software package and manufacturer.
  • Enzyme reactions (for substrate conersion)
    • Methods: provide the final composition and pH of buffer (not necessary to give detailed instructions for preparation);  final concentrations of reactants (for enzymes, provide total units used); final volume; time; temperature. Alternatively, you can provide the various volumes of the reaction components, but you must still provide the concentration and composition of each and the final volume of the reaction.
  • Enzyme reactions (for substrate conversion) with commercial kits
    • Methods: provide the volume and identity of buffer used (state manufacturer), volumes of additives added (e.g. BSA, ATP; state concentration and manufacturer), volume of sample added (state concentration), total volume, time, temperature.
  • Enzyme kinetics data
    • Captions: identify the enzyme, substrate, buffer, and temperature, give the equation of the trendline and the R2 value in the caption.
    • Methods: provide the volume and composition of buffer used (state manufacturer if commercial buffer); final concentrations of all components (e.g. substrate, BSA, ATP); if commecial reagents were used, state volume, stock concentration, and manufacturer for each; if assaying an enzyme solution of unknown composition, provide volume of enzyme solution and state any dilutions that were made; if assaying a known enzyme solution, state either the total units of activity or mass of enzyme added; total volume of the assay; time; temperature; model and manufacturer of instrumentation used to measure (e.g. Thermo Spectronic 20, etc.); identify the parameter that was monitored over time (e.g. wavelength, mass ion); identity of the blank used as the zero reference; describe your methods of data analysis; provide important equations; name the software (package and manufacturer) and explain what features were used; define your activity units.
  • Polyacrylamide gel electrophoresis (PAGE)
    • Captions: percent of acrylamide in gel; staining methodology; identity of lanes; identity of important bands in reference ladder.
    • Methods: percent of acrylamide in gel; composition of the running buffer; approximate amount of sample loaded; type of ladder used including manufacturer; voltage; time; staining procedure with sufficient detail; imaging method (i.e. trans-illumination, epi-illumination ) including wavelength of light.
  • Protein assay (colorimetric methods)
    • Captions: plot the standard curve; identify the protein standard; give the equation of the trendline and the R2 value in the caption.
    • Methods:  State the volumes of reagents, standards, and samples (sample dilution factor, if relevant) used for one assay; identify the protein standard, mass range used (alternatively give stock concentration and volume range used); for reagents, state either the composition or product information (name and manufacturer); the development time and temperature; model and manufacturer of spectrophotometer (e.g. Thermo Spectronic 20, etc.); wavelength(s) measured; identity of the blank used as the zero reference; describe your methods of data analysis (e.g. standard curve plots); explain general process for converting standard curve to a sample concentration; name the software package and manufacturer.
  • Spectrophotometric measurements
    • Model and manufacturer of spectrophotometer (e.g. Thermo Spectronic 20, etc.); wavelength(s) measured; identity of the blank used as the zero reference; dilutions made on sample (if applicable).