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Source information
| Source |
Homo sapience |
| Common nam |
Human |
| Organ |
Kidney |
| Tissue |
Glomerulus |
| Comment |
Glomerulus was highly purified from kidney cotex with no pathologic manifestations obtained from patients under surgical nephrectomy due to renal tumors. |
Experimental conditions
| Sample preparation |
Tissue disruption:
Highly purified glomeruli was homogenized in lysis solution (approx. 40-80 micro L/mg wet tissue) with a Polytron homogenizer for 5 times 15 sec bursts with 45 sec intervals.
Extraction:
15 sec of homogenization repeated 5 times with 45 sec intervals under room temperature. Lysis solution (urea/NP-40/DTT; 9.8M urea, 2% w/v NP-40, 2% Pharmalyte pH3-10, 100mM DTT and 0.01% BPB) used.
(Tsugita, A., et al, Electrophoresis, 21:1853, 2000)
Protease inhibitor:
Conventional PI cocktail-01 (0.5ug/ml E-64, 0.5mM PMSF, 100mM TLCK, 1ug/ml aprotinin, 10ug/ml chymostatin and 0.5mM EDTA).
|
| Protein solution volume |
450(micro L) |
| Protein amount: |
500(micro g) |
| 1st dim. gel name |
Immobiline DryStrip pH3-10 made by Amersham Bioscience |
| 1st dim. equipment |
Amersham Biosciences IPGphore |
| 1st dim. condition |
Rehydration of IPG gel was conducted with rehydration solution prepared by mixing sample extract with rehydration solution (the same composition except that pharmalyte concentration was reduced to 0.5 %)
-Rehydration: 12 hour under 20 degree in C.
-Step1: 500V, 1 hour under 20 degree in C.
-Step2: 1000V, 1 hour under 20 degree in C.
-Step3: 8000V, 10 hour under 20 degree in C. |
| 2nd dim. gel name |
24 cm large format gel |
| 2nd dim. equipment |
Amersham Biosciences Ettan DALT twelve |
| 2nd dim. condition |
Buffer:
6M urea, 30% glycerol, 2% SDS, 50mM Tris-HCl, 50mM DTT and 0.01% BPB.
-Step1: 1W/gel 1hour under 20 degree in C.
-Step2: 20W/gel 4hour under 20 degree in C.
|
| Protein detection |
Silver stain |
| Marker proteins |
Bio-Rad's SDS-PAGE low molecular weight mass standards.
- Trypsin inhibitor, soybean (pI=4.42, MW=21.50)
- Ovalbumin, chicken (pI=4.99, MW=45.10)
- Serum albumin, bovine (pI=5.44, MW=66.20)
- Carbonic anhydrase, bovine (pI=6.46, MW=31.00)
- Egg white lysozyme, chicken (pI=9.22, MW=14.40) |
| Comment |
By using PDQuest, a 2-DE gel image analysis software, valid spots detected in respective 2-DE gel images of glomerular proteins of 4 normal subjects were matched, and a synthetic gel image was created by merging spots in all the gel images. |
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