Antibodies in methods sections

Details to include;

  • Species
  • Clonality (mono/polyclonal/nano)
  • Clone name if appropriate (monoclonal’s usually)
  • Concentration used at (ug/ml rather than dilution where possible)
  • Where you got it from (commercial source or collaborator details).
  • References for where they were generated and validated
  • Don’t forget your secondaries!

If the epitopes matter to your interpretation, include those details too! Ask yourself with this and with other experiments, what details would you need to interpret the data. For example, if you are looking at a transmembrane protein, knowing whether your ab bindings inside or outside the cell could make a difference to interpretation. Or, if you are doing a blot and see processing products then an N-terminal ab could reveal C terminal processing and vice versa. Wherever the details matter, record them!

Antibody validation

So here is a sticky one – do you put your antibody validation data in your results or do you describe them in your methods and then put the data in your supplementary data?

Depends a bit on where the ab came from and therefore how much validation you are describing. If it is published widely, then your validation is probably just your positive and negative controls and they would go in the results (ref the primary description). If it is a relatively new ab and you have done more extensive validation – eg specific experiments to demonstrate specificity – then supplemental data is where I would go.

If you are writing a PhD thesis or dissertation and are relying on an antibody for a key story element then describe your validation in the results section. If I was examining you, I would ask how you proved specificity so you might as well put it straight in!

If you made any antibodies yourself then be as comprehensive as possible; epitope, carrier, boost schedule, primary screening approach, secondary screening, details of hybridoma lines etc and then, definitely, validation figures.

 

Below is an example from one of my papers; as usual, you should aim to do better! (I left out concs)

BaRabbit polyclonal antibodies against ACTN1, ACTN4 and paxillin were purchased from Epitomics Inc. (Burlingame, CA, USA) and rabbit antibodies against lamin A/C were obtained from Cell Signaling (Beverley, MA, USA). Mouse monoclonal antibodies against β4 (3E1), α3 (P1B5) and α6 (6S6) integrin were purchased from Millipore (Billerica, MA, USA) while mouse monoclonal antibodies against talin and vinculin were obtained from Sigma-Aldrich (St Louis, MO, USA). Rabbit polyclonal antibodies against phosphorylated FAK (Tyr397) were purchased from Abcam Inc. (Cambridge, MA). Rhodamine-conjugated phalloidin was purchased from Life Technologies. Monoclonal and polyclonal antibodies against collagen XVII have been described previously (Riddelle et al., 1991). Secondary antibodies conjugated with various fluorochromes or horseradish peroxidase were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).\

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