Dear Laurey,
The problem with basic amino acids disappearing after hydrolysis on PVDF
membranes is not unknown. In a paper from 1989 we wrote:
"For reasons that are unknown to us it appears essential to keep the
PVDF membrane moist during addition of hydrocloric acid, otherwise a
reduction in the recovery of His, Lys and Arg was noted. If the membrane
had been dried it was possible to restore the recovery of the basic
amino acids if the membrane was wet in methanol and rinsed with water
before hydrolysis."
I can add to this, that the methanol should only be 50% in water. If the
membrane is wet with 100% methanol we have seen a loss of methionine,
because it is transformed into homoserine.
The paper is
M. Plough, A.L. Jensen and V. Barkholt
Determination of Amino Acid Compositions and NH2-Terminal Sequences of
Peptides Electroblotted onto PVDF Membranes from Tricine-Sodiumdodecyl
Sulfate-Polyacrylamide Gel Electrophoresis.
Analytical Biochemistry 181 (1989) 33-39.
I hope this solves your problem
Best wishes
Vibeke Barkholt
Department of Biochemistry and Nutrition
Technical University of Denmark
DK 2800 Lyngby, Denmark
+45 4525 2751
Dear Laurey,
The problem with basic amino acids disappearing after hydrolysis on
PVDF membranes is not unknown. In a paper from 1989 we wrote:
"For reasons that are unknown to us it appears essential to keep the
PVDF membrane moist during addition of hydrocloric acid, otherwise a reduction
in the recovery of His, Lys and Arg was noted. If the membrane had been
dried it was possible to restore the recovery of the basic amino acids
if the membrane was wet in methanol and rinsed with water before hydrolysis."
I can add to this, that the methanol should only be 50% in water. If
the membrane is wet with 100% methanol we have seen a loss of methionine,
because it is transformed into homoserine.
The paper is
M. Plough, A.L. Jensen and V. Barkholt
Determination of Amino Acid Compositions and NH2-Terminal Sequences
of Peptides Electroblotted onto PVDF Membranes from Tricine-Sodiumdodecyl
Sulfate-Polyacrylamide Gel Electrophoresis.
Analytical Biochemistry 181 (1989) 33-39.
I hope this solves your problem
Best wishes
Vibeke Barkholt
Department of Biochemistry and Nutrition
Technical University of Denmark
DK 2800 Lyngby, Denmark
+45 4525 2751
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