Printing buffer

Table 1. Spot morphology of oligonucleotides in various print buffers.

Figure 9. Immobilization of antibodies, a) directly on functional beads, or b) via crosslinkers (e.g. biotin-streptavidin chemistry). Arraying onto the slide in c) print buffer or d) entrapped in hydrogel.

Seong (2002) compared silylated (aldehyde) and silanated (amine and epoxy) compounds from several commercial sources to the performance of an antigen (IgG) microarray. In addition, the efficiency of phosphate-buffered saline (PBS) (pH 7.4) and carbonate (pH 9.6) printing buffers were compared. While the various slides and surface chemistries showed differences in their binding isotherms, they ultimately reached similar levels of saturation. Silylated (aldehyde) slides showed comparable loading in both buffer systems. Apparently, tethering of antibody to the surface by Schiff s base formation of the surface aldehyde and lysine residues on the protein was applicable over a broad pH. However, carbonate buffer increased binding of proteins on silanated surfaces. 

Perhaps the least rmderstood factor in the process of microarraying is the print buffer (probe ink) composition. This may not be too much of a surprise because manufacturers of computer printers offer consumers a multitude of different inks (whose formulas are closely guarded trade secrets) for use with a particular printer and kind of paper. In fact, it can be argued that the ink is perhaps the most important piece of the consumable product stream for this manufacturing sector. 

Assume that you are now ready to create your first array on a previously selected substrate. How many elements (spots) do you wish to print This number will determine what kind of pin you will need and while it appears to be a rather fundamental question to ask, it may not be simple to answer. As noted, spot density is directly related to spot size and pitch (Figure 4.25). The pitch will determine how many spots can actually be printed on a slide (Figure 4.26). The pin will deliver a specific droplet volume that will spread to a certain diameter largely based upon the tip s diameter and the print buffer used (Figure 4.27). The larger the spot diameter, the fewer the spots that can be printed (Figure 4.28). 

Print buffer composition should remain simple. For most printing applications, a sodium phosphate buffer at pH 8 to 9 works well. Our laboratory... 

While Diehl et al. (2001) agree that the addition of DMSO to print buffer improves spot uniformity, they argue that DMSO is also toxic and a good solvent for other materials. As a result, they explored alternative chemistries to replace DMSO and also to improve upon postprint blocking conditions in an effort to find a replacement for borate-NMP (l-methyl-2-pyrrolidinone) buffer used for preparing solutions of succinic anhydride for capping of residual amine groups. 

Thus, 3X SSC 1.5 M betaine was used to print down a 500-bp cDNA onto both PLL and aminosilane glass slides and a commercial print buffer, Arraylt microspotting solution or MSS (TeleQrem International, Inc., Sunnyvale, CA) was compared. Curiously, 3X SSC 50% DMSO was not included in this study. While MSS may contain DMSO, the experiments would have been better designed by including 3X SSC + 50% DMSO as a control. 

Nevertheless, it is clear from the work that the inclusion of betaine into the print buffer was an improvement over SSC or the MSS on several fronts. First, the SSC-betaine spotting was formd to increase hybridization efficiency as measured by a 2.5-fold higher hybridization signal intensity for probe-target hybrids relative to those probes spotted in SSC or MSS alone (Figure 4.34). 

McQuain et al. (2003) undertook a detailed study on the effects of relative humidity and a direct comparison of fhe impacts DMSO vs. betaine in print buffer on the overall performance of quill pin printing. A video microscope was employed to visualize and track the drying behaviors of the various printing inks. A Cy5-labeled 466-bp dsDNA probe was used to monitor the printing process. Drop-drying behavior, bulk evaporation from the quill reservoir, surface tension changes, and spothng characteristics (spot diameter, spread, and number deposited) were examined at different RH levels. 

Print buffers 3X SSC, 3X SSC + 50% DMSO, and 3X SSC + 1.5 M betaine were evaluated at 40, 60, and 80% RH for spot intensity, spot diameter, intraspot variation, and CV (Figure 4.35). The reductions in quill drop volumes and droplet drying times were measured by video microscope and the quill reservoir volume changes determined by weight. In summary, "Solvent evaporation from the print buffer reservoir is the major factor responsible for the variations in the transfer of fluid to fhe slide surface."... 

Both 50% DMSO and 3X SSC were about as effective as water as a print buffer in terms of cDNA probe retention ( 20 to 30%), while 3X SSC + 1.5 M betaine retained 60 to 70% of hybridizable probes on the surface. Interestingly, betaine alone appeared to provide better retention than in combination with 3X SSC although the data scatter permits us only to suggest a trend. However, the combination of DMSO and betaine also provided a level of retention comparable to that of betaine ( 70 to +100%) with the added benefit of being able to titrate with DMSO to control spot diameter. Thus, Hessner et al. (2003a) determined that 1.5 M betaine in 3% DMSO provided the optimal print buffer for their studies involving immobilization of cDNA probes onto PLL slides. 

The surfaces included PLL, polystyrene, epoxy-terminated polyethylene glycol (PEG) or dendrimer slides, various amine-derivatized surfaces, and nitrocellulose-coafed slides. All proteins were printed in PBS, rinsed in TBS, and then blocked in 3% nonfat dry milk powder dissolved in TBS-0.1% Tween-20. A final rinse in TBS was performed prior to incubation. While no attempt was made to optimize print buffer or blocking conditions for each of the selected surfaces, if was apparenf fhaf wifh fhe exception of activated polystyrene, most chemistries performed af abouf fhe same levels, i.e., within two- to threefold af saturation. 

Study Print buffer Additives Protein concentration Substrate Rinse Blocking buffer... 

Liu, Y., et al. (2007) Optimization of printing buffer for protein microarrays based on aldehyde-modified glass slides. Front Biosci. 12, 3768-73. 

Prepare crude lysates of each E. coli culture by sonication in P450 Printing buffer and determine the relative expression levels of CYP3A4-BCCP and POR-BCCP by Western blotting. 

Fill up all necessary liquids, including dH O, printing buffer, and wash buffer. Start the printing process (see Note 6). 

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