Characterization of Gel Separated Proteins
85
2
Electroblotting and Direct Sequencer Analysis
The developments in sequencer technology for Edman degradation in recent
years now make it interesting to analyze proteins from 2-D gels directly after
electroblotting to PVDF membranes (Fig. 6.1). Even proteins that stain very
weakly using Coomassie blue, corresponding to 1-2 picomole, can be analyzed.
A problem in this approach is when a blocked N-terminus is encountered.
Although a new separation followed by in-gel digestion can be performed (provided there is more material), deblocking of the protein directly on the membrane after the initial attempt at Edman degradation would increase the efficiency considerably. Since N-terminal blocking is frequently due to an acetyl
group, a protocol for chemical deacetylation (e.g. Gheorghe et aI., 1997) applicable at the low picomole level is an important component to make sequence analysis of proteins electroblotted from 2-D separations more useful (Fig. 6.1).
Sequence analysis of 2-D separated proteins electroblotted to PVDF membranes and stained with Coomassie blue was tested using a human liver microsomal protein fraction isolated via standard techniques including ultracentrifugation at 100000 x g. About 100 !!g of total protein was applied to the first dimension, isoelectric focusing, that was carried out using pH 3-10 non-linear gradient
IPG strips (Pharmacia) for 25 kVh. The second dimension SDS/polyacrylamide
gel electrophoresis employed a 12 % gel and 20 rnA to generate good resolution
in the size range 10-100 kDa. After separation- and staining, several protein containing spots were cut from the PVDF membrane and applied to Procise cLC
sequencer analysis. One of the excised spots revealed 70 femtomole of glutamic
acid in the first cycle (Table 6.1) with a PTH signal that was roughly 5-fold above
Table 6.1. N-terminal
Cycle
Residue
Amount recovered, femtomole
sequences analysis of human
78 kDa glucose regulated pro(background orrected)
tein electroblotted onto PVDF
Glu
70
membrane after 2-D separation
of a liver microsomal protein
2
Glu
100
fraction
3
Glu
20
4
Asp
100
5
Lys
120
6
Lys
140
7
Glu
100
8
Asp
50
9
Val
70
10
Gly
50
11
Thr
40
12
Val
10
85
2
Electroblotting and Direct Sequencer Analysis
The developments in sequencer technology for Edman degradation in recent
years now make it interesting to analyze proteins from 2-D gels directly after
electroblotting to PVDF membranes (Fig. 6.1). Even proteins that stain very
weakly using Coomassie blue, corresponding to 1-2 picomole, can be analyzed.
A problem in this approach is when a blocked N-terminus is encountered.
Although a new separation followed by in-gel digestion can be performed (provided there is more material), deblocking of the protein directly on the membrane after the initial attempt at Edman degradation would increase the efficiency considerably. Since N-terminal blocking is frequently due to an acetyl
group, a protocol for chemical deacetylation (e.g. Gheorghe et aI., 1997) applicable at the low picomole level is an important component to make sequence analysis of proteins electroblotted from 2-D separations more useful (Fig. 6.1).
Sequence analysis of 2-D separated proteins electroblotted to PVDF membranes and stained with Coomassie blue was tested using a human liver microsomal protein fraction isolated via standard techniques including ultracentrifugation at 100000 x g. About 100 !!g of total protein was applied to the first dimension, isoelectric focusing, that was carried out using pH 3-10 non-linear gradient
IPG strips (Pharmacia) for 25 kVh. The second dimension SDS/polyacrylamide
gel electrophoresis employed a 12 % gel and 20 rnA to generate good resolution
in the size range 10-100 kDa. After separation- and staining, several protein containing spots were cut from the PVDF membrane and applied to Procise cLC
sequencer analysis. One of the excised spots revealed 70 femtomole of glutamic
acid in the first cycle (Table 6.1) with a PTH signal that was roughly 5-fold above
Table 6.1. N-terminal
Cycle
Residue
Amount recovered, femtomole
sequences analysis of human
78 kDa glucose regulated pro(background orrected)
tein electroblotted onto PVDF
Glu
70
membrane after 2-D separation
of a liver microsomal protein
2
Glu
100
fraction
3
Glu
20
4
Asp
100
5
Lys
120
6
Lys
140
7
Glu
100
8
Asp
50
9
Val
70
10
Gly
50
11
Thr
40
12
Val
10