Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3

M Ploug; A L Jensen; V. Barkholt

Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3

146 Citationer (Scopus)

Abstract

The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Schägger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.

Originalsprog	Engelsk
Tidsskrift	Analytical Biochemistry
Vol/bind	181
Udgave nummer	1
Sider (fra-til)	33-9
Antal sider	7
ISSN	0003-2697
Status	Udgivet - 15 aug. 1989
Udgivet eksternt	Ja

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Citationsformater

Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3. / Ploug, M ; Jensen, A L; Barkholt, V.
I: Analytical Biochemistry, Bind 181, Nr. 1, 15.08.1989, s. 33-9.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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abstract = "The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Sch{\"a}gger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.",

keywords = "Amino Acid Sequence, Amino Acids, Collodion, Complement C3, Electrochemistry, Electrophoresis, Polyacrylamide Gel, Glycine, Membranes, Artificial, Peptide Mapping, Peptides, Polyvinyls, Sodium Dodecyl Sulfate, Journal Article",

author = "M Ploug and Jensen, {A L} and V. Barkholt",

year = "1989",

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T1 - Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

T2 - application to peptide mapping of human complement component C3

AU - Ploug, M

AU - Jensen, A L

AU - Barkholt, V.

PY - 1989/8/15

Y1 - 1989/8/15

N2 - The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Schägger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.

AB - The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Schägger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.

KW - Amino Acid Sequence

KW - Amino Acids

KW - Collodion

KW - Complement C3

KW - Electrochemistry

KW - Electrophoresis, Polyacrylamide Gel

KW - Glycine

KW - Membranes, Artificial

KW - Peptide Mapping

KW - Peptides

KW - Polyvinyls

KW - Sodium Dodecyl Sulfate

KW - Journal Article

M3 - Journal article

C2 - 2817381

SN - 0003-2697

VL - 181

SP - 33

EP - 39

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 1

ER -