To reduce non-specific reactions and gain a higher sensitivity, high molecular weight proteins contained in the sera were removed by Multiple Affinity Removal Spin Cartridge Reagent Kit (Agilent Technologies, Santa Clara, CA, USA) and used for the serum samples.
Excess polymer was removed by multiple centrifugation and resuspension in ultrapure water.
Excess paraffin was removed by multiple washes in xlyene.
Unbound protein was removed by multiple washes with binding buffer and bound protein was eluted in 100 µl elution buffer (10 mM glutathione, 50 mM Trizma-HCl, pH 7.4) at room temperature for 30 minutes.
This bias was removed by multiple imputation.
Residual trifluoroacetic acid (TFA) from peptide synthesis was removed by multiple rounds of lyophilization against a 0.1 M DCl solution.
As the mice were part of dietary study, the effect of diet as well as of sex was removed by multiple regression of these variables on the phenotypic traits and addition of the residuals to the trait means.
After 4 h of incubation at 37°C in modified Medium199, non-phagocytosed parasites were removed by multiple washing steps with PBS and incubation was continued for another 20 or 44 h in IMDM at 37°C and 9% CO2.
Unbound proteins and non-specific contaminants were removed by multiple washes of buffer C alone followed by sequential single washes of 1 m KCl, 0.05% Triton X-100, 1 m m ATP and 40 m m imidazole contained in buffer C. Finally, the bound-proteins were eluted with buffer C containing 250 m m imidazole, and the samples were dialyzed against appropriate buffers for use, in particular experiments.
