Writing about immunoblotting

How western blots / immunoblots methods and data are written about has come under a lot of scrutiny recently and new guides to best practice are being implemented in all the reputable journals. This is turn means that the standards reviewers/examiners will expect will be higher. This is a good thing, it means that the papers you read now will be better but it also means you must hold yourself to these higher standards if you want to be taken seriously.

As always, it’s the details that matter. Here are things you should always report:

  • Sample preparation; buffer, volumes/mass or volume/area or volume/cell, times to extraction, etc must be fully detailed
  • Running, gel and transfer conditions; gel type and %, buffers, transfer type and duration, membrane type, pore size and brand. Native/reduced.
  • Blocking solution, time and amount
  • Antibodies – primary, secondary, catalog numbers, supplier, lot number, concentration (see note on antibodies –  *I usually write abs as a separate methods sub-section if I use them for more than one experiment type.
  • Incubation times and temperatures for both antibody steps and in what buffers
  • Washing solution, time, how often
  • Detection reagents and imaging techniques, scan times/laser intensities etc including brand of X-ray film
  • Molecular mass of band of interest – which markers did you use (indicate marker location on blot). Most good journals will require an uncropped blot to be included as supplemental figures.
  • Balancing loading – If you intend to quantify your immunoblots you must clearly explain how protein loading was normalised between lanes.
    • Normalisation to total protein loading is preferred but which total protein stain?
    • If you intend to use a reference protein for quantification/to balance loading you need to provide the evidence reference protein expression did not change with your experimental conditions
  • Quantification
    • Your methods should detail how the linear range for your antibodies were determined (prepare that figure for inclusion as a supplemental fig)
    • Signals obtained using antibodies to phosphorylated epitopes should be normalised to total protein level of the target protein
    • Software used
    • How! compared what to what, plotted as what
  • Replicates – technical and biological repeats numbers and how you defined independence (i.e. what you count as a biological repeat) must be defined
  • Statistics – as always, what tests, what comparisons, what threshold did you decide to accept as significant?

Use these bullets as a checklist and you will do fine!

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