I found in several articles where the authors use the quantitative analysis (densitometric) of the WB bands, where in most cases the significant data is not found. I think that obtaining significant densitometric data for bands is very difficult due to the difference between the intensity of the individual experimental data. At least, I have done an experiment several times, and I did not find the densitometric data differed significantly. I used LAS1000 (Fuji Film) for the analysis.
Now my question- is it really important to include this statistical analysis in the paper?
Can anyone suggest some recent papers (especially with/without the densitometric analysis of the phosphorylated protein), or give some important advice regarding this?
Thank you!
In general, a semi-quantitative visualization of Western blot results is absolutely value-free. This is - amongst other things - due to non-standarized normalization of results (pictures, differend controls, standards etc.), subjectively interpretation of findings on "pictures" of more or less sufficient quality, limited comparability of results (e.g. due to utilization of different methods/protocolls, apparatures, sample preparation, exposure parameters ...) and so on. Moreover, there is a great impact of the experimentator self (methodological routine, subjective vs objective data interpretation etc.).
Thus, it is sufficient to present Western blots results (pictures) and to interprete these data as they are. Semi-quantitative and non-informative as well as rarely
verifiable not-objective presentation of more or less reliable pseudo-quantitative "valures"/"figures" is neither helpful nor useful in this context.
It is important to use densitometric analysis as a more objective parameter in interpreting your results/bands. Western blots have been notorious for researchers falsifying data. The visual impression - which many times is difficult to interpret due to loading inequalities, contrast exposure or very faint bands - should be corrobarated by densitometric analysis.
I don't think so. Western blot results are an absolutely subjective type of data. Controls as well as other nomalization parameters differ between labs as well as between different experimentators in one lab. Also method for normalization for visualization of results via pseudo quantification are often completely different between different labs. Thus, the obtained results will differ in every experimental set from data quantified by others. To offer quantitative data a more reliable and adequate method should be used, such as ELISA.
Western blot results will be accepted by every journal without quantification, since this is a more "uncertain" source of results. Such data presentation often rather leads to artefacts than to "real qualitative findings". The obtained results are neverever comparable when two different labs evaluate the same visual results of a Western blot experiment. Thus, results presented as "visual quantification" may differ from "real results obtained by (completely) qualitative Western blot results.
Hi Syed,
If you have already sent the paper, and the reviewer now ask you to put the quantifications, just do it, there is no worth to fight for it.
When your antibody is very tricky and the results were not stable, and there is no other choice to present the same data, you should repeat WB, and use normalized values or fold increase or whatever, and when the averaged results is enough convincing, for that purpose, the measurements of density is not a bad choice.
When you would like to see the phosphorylations or the degradation of the particular band, the quantification is quite informative.
Good Luck your experiments
Thank you Andreas Eisenreich , Sei Kuriyama and Ioasaf Karafotias for your important advice.
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