Journal of Clinical Investigation -- All data are not created...

For the examples in this editorial, let’s discuss Western blots in particular (though the rules apply to Northern, Southern, and PCR blots too).

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bands can be spliced together, but only, and I repeat only, if they were noncontiguous but run on the same gel at the same time

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a thin line in between the spliced lanes and appropriate text added to the figure legend to reflect the modification

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there is nothing more reliable than a blot with bubbles — there is no need to erase background noise or doublet bands

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My understanding of a loading control is that it represents an analysis of an irrelevant protein from the exact same gel lane to assess how much sample was loaded in that particular lane.

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a loading control, say β-actin or GAPDH, was probed off the same gel

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elementary governing principle to me

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an article that, among other problems, ran the loading control on a separate gel at the same time

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running a parallel gel, even if the sample run on the gel was an aliquot from the same tube, does not demonstrate equal loading of sample in the experimental gel

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How does one control for variations in pipetting such small volumes?

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can’t you just cut off the bottom and reprobe that part for the actin or other loading control and reprobe the top for another protein of interest?

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some blots need to be stripped and reprobed several times

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run duplicate blots and present 2 rows of loading controls?

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worried about the manipulations we can detect

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a heavy (or light) hand with a pipette can influence a band’s appearance when no loading control is there to normalize it

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in a particular row of a Western, all bands presented should be from the same exposure time

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exposure times

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allow band splicing — with all the appropriate caveats, as described

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splicing lanes from various exposures of the same blot doesn’t prove anything

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expose most any gel long enough, you’ll get bands to appear and the results you want

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can always add another row of lanes to your figure that show a longer exposure time to verify that the protein was there

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keep the raw data

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A well-annotated lab notebook can resolve problems very quickly.

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Scan the film from your blot and save it on your computer and on the lab server, and keep a copy on a USB drive or elsewhere

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data were on a single USB drive that had been lost

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paste the film into your lab notebook

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Label the film clearly and annotate the date and conditions.

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Do not lose it when you move your lab

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protect the lab notebook

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could not substantiate the data

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e-mail a scan of the uncut, labeled film to yourself and the senior author to ensure the data are accessible

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Label the data

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multiple copies

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print them

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clear records

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If you are the senior author, it is incumbent on you to verify all of the raw data yourself.

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There is intense pressure to produce, and to produce high-impact results.

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doing anything to please

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you should be able to verify every single piece of data in it and take responsibility

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tools to be able to detect whether you have altered your figures in any way

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on 2009-03-28 by amiigo

These principles could be used in cooperation with authors to help them ensure accurate data records are preserved.

I hope that the experiments themselves in our published papers have been performed properly, but that is not something we can police.

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on 2009-03-28 by amiigo

The processes of peer review and reproduction of experimental results works together with careful examination and preservation of published material.

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on 2009-03-28 by amiigo

on 2009-03-28 by amiigo

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