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ConclusionsĬollectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB 1 receptor expression, and potentially of any GPCR, in a variety of biological samples. Estimated values of CB 1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443–473 of the mouse CB 1 receptor that corresponds to residues 442–472 in the human homolog. Here we generated highly soluble and stable recombinant protein constructs GST-CB1 414–472 and GST-CB1 414-442 containing much of the human CB 1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB 1 receptor expression on crude synaptosomes of the adult rat brain cortex. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. GPCRs in general and cannabinoid CB 1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. In the end, the integrity and robustness of the published scientific data should be part of the considerations while planning image analysis experiments.Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. Sometimes, a qualitative interpretation might be better suitable than a flawed quantification. IMHO: Not because we can measure everything means also we should do it. I can only recommend to use either the software of the Western Blot imaging system in the lab or alternatively GelAnalyzer because it allows to stick pretty well to the recommended procedure by Western Blot material suppliers (which I would guess are the most experienced people in that particular field) and as seen in the publication above. Hammond, “A defined methodology for reliable quantification of Western blot data.,” Mol. So, generally there are many pitfalls related to WB measurements.īesides the literature list in one of the linked posts above, mainly the following gives a good insight into the procedure: And band selections which overshoot the actual band also lead to wrong results. One cannot reliably quantify bands by making boxes around them and using analyze measure nor do horizontal lines fulfill the requirements. Here is one video which is explaining a little more detailed the considerations of WB in general, normalization as well as measurements The latter and pretty much of most other ones completely ignore every thing mentioned in scientific literature regarding Western Blot measurements (and the pre-requisites for it). There are many videos online like the linked one above.
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But I cannot contain myself to add my 2 cents to this topic, because some of those videos make me like… No offense to no one making those videos or taking them as orientation in case of the lack of other available resources. This was the one I looked at Analysing blots and gels with ImageJ/Fiji - YouTube