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Table 1 Comparison of spot parameters in IPG- and NEPHGE-based 2DE at standard protein load

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

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

102

79

23

110

80

30

Reproducibility of spots6%

75 ± 4%

82 ± 1%

44 ± 18%

76 ± 17%

72 ± 18%

87 ± 20%

Total7 protein volume in a gel (Vol)

410302 ± 76913

325075 ± 73300

85227 ± 13613

444930 ± 75631

278277 ± 69205

166653 ± 15835

(100 ± 19%)

(79 ± 4%)

(21 ± 4%)

(100 ± 17%)

(63 ± 6%)

(37 ± 6%)

Variation of spot volume (ΔVol)8

35% ± 25

36% ± 25

30% ± 25

40% ± 33

46% ± 36

27% ± 21

Variation in relative volumes of spots (Δ%Vol)9

30% ± 23

30% ± 23

26% ± 19

31% ± 28

31% ± 30

31% ± 25

Average saliency of protein spot10

2794 ± 293

2927 ± 307

2248 ± 947

2610 ± 549

2202 ± 558

3523 ± 810

Low quality spots (saliency <500)11%

15 ± 3%

14 ± 6%

21 ± 11%

27 ± 8%

30 ± 9%

20 ± 9%

  1. 1The same samples were analysed in IPG- and NEPHGE-based 2DE systems; ~50 μg of whole cell protein was loaded onto IPG strips and ~30 μg onto NEPHGE gels (due to small space for sample application in NEPHGE tubes – see text).
  2. 2Immobilized pH gradient (IPG) based 2DE method (Invitrogen pH3-10 system).
  3. 3 Non-equilibrium pH gradient gel electrophoresis (NEPHGE) based 2DE method (WITAvision pH3-10 system).
  4. 4Parameters were calculated from 2–3 replicas (repeating analysis of the same samples). Each parameter was calculated both for whole gel (pI 3–10, all detected proteins) and for its pI <7 and pI >7 parts (acidic and basic proteins, respectively). Neutral pI 7, separating acidic and basic protein spots, is indicated by dashed line in Figure 1.
  5. 5Number of detected separate protein spots in all samples (Control, MeH ir MeN), from all replicas.
  6. 6The same spots detected among replicas of the same sample (according to matches of the spots generated by 2D image analysis software ImageMaster 2D Platinum 7.0); the percentage of matched spots (±SD) is given for a whole gel (pI 3–10) and for its acidic or basic parts.
  7. 7Total volume (Vol; product of spot area and intensity) of all protein spots in one gel is calculated by 2D analysis software; here the average from whole pI3-10 gel is given as 100% (±SD), whereas pI <7 and >7 indicate acidic and basic protein portions, respectively.
  8. 8Variation of volumes of the same spots in separate replicas; calculation was made using all spots matched by the software and then the average of variation ΔVol ±SD was calculated.
  9. 9%Vol indicates percentage of volumes of separate spots among volume of all protein spots in a gel. In this case, all matched protein spots were evaluated in the same way as calculating variation of volumes (8), only instead Vol the values of %Vol was used (the result is average of Δ%Vol ± SD).
  10. 10Average saliency of detected protein spots per gel ± SD.
  11. 11Detected protein spots with saliency <500 were considered as low quality spots (see text). The percentage of such protein spots (±SD) was calculated for a whole gel (pI 3–10) and for its acidic or basic parts.
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