Glycine, threonine, arginine, glutamate, and histidine were used as buffer components in the equilibration, washing, and elution steps of these chromatographies.
However, further studies have to be undertaken to identify the protein buffer components in the human salivary proteome.
Separation parameters such as injection time, buffer components, pH of the buffer were studied.
(PBS buffer components used in this paper: 10 mM phosphate, pH 7.4, 138 mM NaCl, 2.7 mM KCl, and 2 mM MgCl2).
The buffer components in a gel-MMV plate (1 × SSC) were exchanged with LB broth and the affluent solution in wells was removed by centrifugation.
The magnetic separation plate was inserted into the hand-held magnetic plate washer and the plate was washed three times using 100 μl wash buffer (wash buffer components 1 and 2 in nuclease-free water) per well.
To analyse the potential masking effect of specific buffer components in more detail, we diluted known amounts of E. coli LPS 055 B5 in three different masking buffers containing a) 10 mM sodium citrate and 0.05% Tween-20, b) 10 mM sodium citrate and 10 mg/ml BSA, and c) 10 mM sodium citrate and 0.05% Triton X-100.
The mix was optimized for real-time PCR analysis using SYBR Green 1 Dye, AmpliTaq Gold DNA polymerase, dNTPs with UTP, Passive Reference 1 required for signal normalization, and optimized buffer components, in a total volume of 50 μL.
Amplification of each locus was performed in a total volume of 10 μl, containing 0.25 μM of each primer, 2 μl genomic DNA and 2× PCR Master Mix (Applied Biosystems master mix contains AmpliTaq Gold® DNA Polymerase, Ultra Pure water, deoxyribonucleotide triphosphates (dNTPs), ROX™ Passive Reference and Buffer components) in a programmable thermocycler (Applied Biosystems PRISM 7900HT®).
Tris buffer was used for these assays to minimize the background signal in the HPLC chromatograms that arose due to buffer components.
The peptide fragments formed during digestion were concentrated on an RP trapping column (TC) and salts and other buffer components present in the sample were removed.
