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Typus
Verschleierung
Bearbeiter
Graf Isolan
Gesichtet
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Untersuchte Arbeit:
Seite: 44, Zeilen: 9-31
Quelle: Zibrova 2004
Seite(n): 48, 63-64, Zeilen: 48:7-10; 63:13-15.21-32 - 64:1-3
Aliquots of prepared tissue homogenates and cell lysates were denatured in sample buffer containing 2% SDS, 10% glycerol, 50 μg/ml bromphenol blue, 2% β- mercaptoethanol and 50 mM Tris-HCl, pH 6.8 by boiling at 95°C for 10 min and 15 μg of total protein was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

3.3.2.b SDS-polyacrylamide gel

For all applications described, a 12.5% separating Tris/glycine SDS polyacrylamide ready made gel (SDS-PAGE) was used from invitrogen as instructed. The western blot was performed according to the method of Laemmli (Laemmli 1970).

3.3.2.c SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic transfer

The samples were loaded onto the bottom of the wells. Electrophoresis was run at constant 20 mA per gel. The Rainbow™ colored protein markers (Amersham Pharmacia Biotech) were used as molecular weight standards. Electrophoretic transfer was carried out essentially as described by Towbin (Towbin et al. 1979). Prior to stopping the gel running, fiber pads, filter paper and nitrocellulose transfer membrane (0.45 μM pore size) were soaked in transfer buffer. After electrophoresis, the gel was removed out of the plates and immersed in transfer buffer. For electrophoretic transfer of proteins from the gel to a membrane, a Mini-Trans-Blot® Cell (Bio-Rad), compatible with described system for electrophoresis, was utilized. The transblot sandwich was assembled according to the manufacturer’s instructions from Bio-Rad in the following order starting from the anode side: sponge, 2 sheets of filter paper, nitrocellulose membrane, gel, 2 sheets of filter paper, sponge. The assembled transblot sandwich was inserted into the transblot cell filled with transfer buffer. Ice-cooling unit was set behind the cathode side of transblot cell. The transfer ran for 2 h at 350 mA with one change of the ice-cooling unit after the first hour.


Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685

Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350-4354

[Page 48]

Aliquots of the prepared lysates were denatured in sample buffer containing 2% β-mercaptoethanol by boiling at 95°C for 5 minutes and 20-60 μg of total protein was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[Page 63]

Casting of SDS-polyacrylamide gel

For all applications described, a Tris/glycine SDS polyacrylamide gel (SDS-PAAG) system was used according to the method of Laemmli (Laemmli, 1970). [...]

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic transfer

The samples were loaded onto the bottom of the wells. Electrophoresis was run at a constant current of 25 mA per gel. Electrophoretic transfer was carried out essentially as described by Towbin (Towbin et al., 1979). Prior to stopping the gel running, fiber pads, filter paper and nitrocellulose transfer membrane (0.45 μM pore size) were soaked in transfer buffer. After electrophoresis, the gel was removed out of the plates and immersed in transfer buffer. For electrophoretic transfer of proteins from the gel to a membrane, a Mini-Trans-Blot® Cell (BioRad), compatible with the described system for electrophoresis, was utilized. The transblot sandwich was assembled according to the manufacturer’s instructions from BioRad in the following order starting from the anode side: sponge, 2 sheets of filter paper, nitrocellulose membrane, gel, 2 sheets of filter paper, sponge. The assembled transblot sandwich was inserted into the transblot cell

[Page 64]

filled with transfer buffer. An ice-cooling unit was set behind the cathode side of transblot cell. For most of the proteins, the transfer ran for 2 hours at 500 mA with one change of the ice-cooling unit after the first hour.


40. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685, 1970.

90. Towbin H., Staehelin T., and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc.Natl.Acad.Sci.U.S.A. 76: 4350-4354, 1979.

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(Graf Isolan) Schumann

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