Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae

Table 2 Comparison of spot parameters in IPG- and NEPHGE-based 2DE at high protein load

Method ¹	IPG ²			NEPHGE ³
Parameter ⁴	pI 3-10	pI <7	pI >7	pI 3-10	pI <7	pI >7
Number of detected protein spots⁵	432	321	111	506	372	134
Reproducibility of spots⁶%	68 ± 1%	73 ± 4%	51 ± 13%	87 ± 5%	85 ± 6%	90 ± 4%
Total⁷ protein volume in a gel (Vol)	1726878 ± 260176	1357575 ± 226314	369303 ± 59726	2618475 ± 58090	1845417 ± 54127	773057 ± 30071
Total⁷ protein volume in a gel (Vol)	(100 ± 15%)	(79 ± 3%)	(21 ± 3%)	(100 ± 2%)	(70 ± 1%)	(30 ± 1%)
Variation of spot volume (ΔVol)⁸	49% ± 55	48% ± 54	55% ± 58	26% ± 31	28% ± 34	22% ± 23
Variation in relative volumes of spots (Δ%Vol)⁹	47% ± 51	46% ± 50	53% ± 57	25% ± 31	27% ± 34	21% ± 22
Average saliency of protein spot¹⁰	1931 ± 348	2028 ± 366	1419 ± 348	3210 ± 136	2947 ± 307	4189 ± 205
Low quality spots (saliency <500)¹¹%	20±5%	18±5%	28±7%	11±4%	13±5%	6±3%

2x higher protein amounts were loaded onto 1st dimension gels, than for standard application described in Table 1. Preparation of concentrated whole cell lysates for this experiment is described in Methods section. Other procedures and all calculations were the same as for 1x protein load described in Table 1. An example of 2D gel images from a high load experiment is shown in Figure 2.
¹The same samples were analysed in IPG- and NEPHGE-based 2DE systems; the equal amounts of ~100 μg of whole cell protein were loaded onto IPG strips and onto NEPHGE gels.
^2–11The references are the same as in Table 1 and all parameters were calculated exactly as described in Table 1 legend.

ISSN: 1477-5956