Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics
Table 1
Summary of protein extraction conditions. Yield was calculated as the total µg of protein divided by the area of the tissue section multiplied by the section thickness of 4 µm.
Control
(A)
(B)
(C)
(D)
(E)
(F)
Qkit
Extraction buffer
7 M urea 2 M thiourea 1 M AmBic
Same as control
7 M urea 2 M thiourea 2 M AmBic pH 7.2
7 M urea 2 M thiourea 1 M AmBic 0.2% Zwittergent 3-16
Same as control
EXB plus extraction buffer
First temperature
95°C 1.5 hr
N/A
95°C 20 min
100°C 20 min
Second temperature
N/A
60°C 2 hr
80°C 2 hr
60°C 2 hr
80°C 2 hr
Trypsin digestion
Overnight 37°C 1 : 20 (w/w)
30 min 50°C 1 : 5 (w/w)
Overnight 37°C 1 : 20 (w/w)
Total protein (µg)
19.4
26.3
40.6
34.4
30.6
41.0
Protein yield (µg/mm3)
2.94
3.99
6.16
5.22
4.63
6.03
4.97
6.22
Protein extracted from a single section of tissue.
