Each strip was then incubated at 4°C, O.N. with either a commercial antibody or a serum sample diluted in dilution buffer (TBS, 0.5% western blotting reagent and 0.02% sodium azide).
A thick band between the supernatant and Percoll fraction was collected for each sample, diluted with dilution buffer (5 mM HEPES, pH 7.4, 3 mM CaCl2, 1 mM EGTA), and centrifuged at 14,000 rpm for 30 min at 4°C.
Sample wells were prepared by adding 10 μl of a 100-fold dilution of each sample (diluted in antioxidant assay buffer), 10 μl of metmyoglobin, and 150 μl of chromogen.
After optimization of all the significant variables and interactions, the recommended procedure was established as follows: DSPME (using a polydimethylsiloxane (PDMS)/divinylbenzene (DVB) coating) of 1 mL of milk sample diluted with Milli-Q water (1 10 dilution ratio), at 100 °C, under stirring for 30 min.
The myosin sample, diluted to ∼0.5 or 2 mg/ml in their respective dilution buffer, were applied to copper grids coated with Quantifoil holey carbon films (Quantifoil Micro Tools GmbH, Jena, Germany) for 30 s, and replaced by an additional drop of sample (30 s).
The pure recombinant proteins were incubated with the plasma sample diluted 1 400 overnight, centrifuged and applied to the membrane blot containing fetal monkey brain.
The best overall analytical conditions obtained were the following: 35 mL of sample, diluted 1 1 with Milli-Q water and extraction at 1100 rpm for 100 min.
That can require a series of assays to be performed on a sample diluted to different strengths, in order to assemble a complete picture of a protein's concentration in the matrix.
FASI basically consists in a mismatch between the electric conductivity of the sample and that of the running buffer and it is achieved by electrokinetically injecting the sample diluted in a solvent of lower conductivity than that of the carrier electrolyte.
The sample, diluted to 0.45 mg/mL with PBS, pH 7.4, was heated from 20 to 90 °C at a scanning rate of 60 °C/h.
The pH of the substrates was measured by utilizing 5 g of the sample diluted in 50 ml of distilled water.
