PMC:4996381 / 29373-30947 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996381","sourcedb":"PMC","sourceid":"4996381","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996381","text":"2.6. Quantification and Visualization\nPierce® ECL Western blotting Substrate (Thermo Scientific, distributed by Fisher Scientific, #32106) was used to reveal the bound antibody and chemiluminescence was detected with an image reader (Fujifilm LAS-4000 ver.2.0, GE Healthcare Life Sciences) typically using an incremental exposure time of 5 min for a total of 80 min (or until saturation was reached, in the case of very strong signal). The resulting SPOT intensities were quantified with the Kodak 1D V.3.6.2 Scientific Imaging System. Two profiles were generated for each SPOT, one covering the outer boundary of each SPOT (large profile) and one smaller (co-centric to the large profile) covering ~50% of the large profile (small profile). Numeric values were imported in Microsoft Excel, profiles were averaged and the intensity was normalized throughout the membrane between 0 and 100. We found that we could identify “corona” effects when the smaller profile intensity was 80%–85% of the large profile intensity, requiring manual adjustment of the data and further experiments. Data were binned using arbitrary derived values for each set of experiments by visual inspection of the membranes resulting in classification of SPOTs as “weak” (typically for normalized intensity values between 5% and 20%), “medium” (typically for normalized intensity values between 20% and 60%) and “strong”(typically for normalized values between 60% and 100%). Visualization was carried out with a simple VBScript within Microsoft Excel taking into account the intensity bins described.","divisions":[{"label":"Title","span":{"begin":0,"end":37}}],"tracks":[]}