Role of amyloid beta in brains of people with Alzheimer's under scanner [newKerala.com News # 644]:
'via Blog this'
Saturday, March 31, 2012
Monday, March 19, 2012
Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS : Article : Nature Protocols
Use of Nitrocellulose Membranes for Protein Characterization by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
We present an improved method for MALDI-MS analysis
of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted
proteins can be analyzed directly for intact molecular
weight determination or after on-membrane digestion by
dissolution of the nitrocellulose in MALDI matrix solution
containing 70% acetonitrile and 30% methanol. This
solution helps maintain solubility of proteins and peptides
while dissolving the NC membrane, which is dissolved
by 100% acetone in other protocols. On-membrane tryptic
digestion using this method requires half the time of ingel digestion and results in fewer missed cleavages and
better protein coverage. For the membrane proteins
studied, bovine uroplakins II and III, the protein coverage
was almost twice that provided by conventional in-gel
digestion, and the transmembrane domains of both
uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDIMS of a new dye, MemCode, which is specifically designed
for staining NC membrane-immobilized proteins and is
faster and more sensitive than Ponceau-S. Our improved
on-membrane digestion protocol greatly improves the
study of soluble and, particularly strikingly, integral
membrane proteins by mass spectrometry http://webdoc.nyumc.org/nyumc/files/sun-lab/attachments/2006.AC.MSa.pdf
'via Blog this'
of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted
proteins can be analyzed directly for intact molecular
weight determination or after on-membrane digestion by
dissolution of the nitrocellulose in MALDI matrix solution
containing 70% acetonitrile and 30% methanol. This
solution helps maintain solubility of proteins and peptides
while dissolving the NC membrane, which is dissolved
by 100% acetone in other protocols. On-membrane tryptic
digestion using this method requires half the time of ingel digestion and results in fewer missed cleavages and
better protein coverage. For the membrane proteins
studied, bovine uroplakins II and III, the protein coverage
was almost twice that provided by conventional in-gel
digestion, and the transmembrane domains of both
uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDIMS of a new dye, MemCode, which is specifically designed
for staining NC membrane-immobilized proteins and is
faster and more sensitive than Ponceau-S. Our improved
on-membrane digestion protocol greatly improves the
study of soluble and, particularly strikingly, integral
membrane proteins by mass spectrometry http://webdoc.nyumc.org/nyumc/files/sun-lab/attachments/2006.AC.MSa.pdf
'via Blog this'
Mass spectrometric characterization of proteins transferred from polyacrylamide gels to membrane filters - Ino - 2011 - FEBS Journal - Wiley Online Library
У сацыяльных сетках выберуць лепшую беларускую прозу: Агенцтва рэгіянальнага развіцця “Дзедзіч” у межах кампаніі “Наша мова” адкрывае галасаванне за найлепшую прозу на беларускай мове.
больш па тэме
Аудио:
Analysis of Electroblotted Proteins by Mass Spectrometry: Protein Identification after Western Blotting
We describe a new approach for the identification and characterization by mass spectrometry of
proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting And
Removal of Nitrocellulose, or BARN), proteins can be analyzed either as intact proteins for
molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is
used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus
removing the nitrocellulose which can interfere with mass spectrometry analysis. This method
offers improved protein coverage, especially for membrane proteins such as uroplakins, since the
extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the
sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass
spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble
and membrane proteins after Western blotting, obtaining comparable or better results than with
in-gel digestion.
http://www.mcponline.org/content/early/2007/10/15/mcp.M700415-MCP200.full.pdf
proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting And
Removal of Nitrocellulose, or BARN), proteins can be analyzed either as intact proteins for
molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is
used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus
removing the nitrocellulose which can interfere with mass spectrometry analysis. This method
offers improved protein coverage, especially for membrane proteins such as uroplakins, since the
extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the
sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass
spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble
and membrane proteins after Western blotting, obtaining comparable or better results than with
in-gel digestion.
http://www.mcponline.org/content/early/2007/10/15/mcp.M700415-MCP200.full.pdf
Internal amino acid sequence analysis of proteins separated by oneor two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose
http://www.pnas.org/content/84/20/6970.full.pdf
We have developed a general two-step method for obtaining peptide fragments for sequence analysis from
picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out,
and each protein is digested in situ by proteolytic enzymes such
as trypsin or staphylococcal V-8 protease. The resulting peptide
fragments are separated by narrow-bore reverse-phase HPLC,
collected, and sequenced in a gas-phase sequenator. Excellent
peptide recoveries and the absence of extraneous contaminants
in the separation of the peptide fragment mixture allow the
generation of extensive internal sequence information from
picomole amounts of protein. The method thus overcomes the
problem of obtaining amino acid sequence data from Nterminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate
oligonucleotide probes for molecular cloning and/or used to
search sequence data bases for related proteins. This method
has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from oneand two-dimensional polyacrylamide gels.
'via Blog this'
We have developed a general two-step method for obtaining peptide fragments for sequence analysis from
picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out,
and each protein is digested in situ by proteolytic enzymes such
as trypsin or staphylococcal V-8 protease. The resulting peptide
fragments are separated by narrow-bore reverse-phase HPLC,
collected, and sequenced in a gas-phase sequenator. Excellent
peptide recoveries and the absence of extraneous contaminants
in the separation of the peptide fragment mixture allow the
generation of extensive internal sequence information from
picomole amounts of protein. The method thus overcomes the
problem of obtaining amino acid sequence data from Nterminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate
oligonucleotide probes for molecular cloning and/or used to
search sequence data bases for related proteins. This method
has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from oneand two-dimensional polyacrylamide gels.
'via Blog this'
On-Membrane Tryptic Digestion of Proteins for Mass Spectrometry Analysis
Identification of proteins and characterization of posttranslational modifications are crucial steps for
many biological, biochemical, and biomedical studies, and mass spectrometry has become the method of
choice for these analyses. Here we describe two methods for the on-membrane digestion of proteins electroblotted onto nitrocellulose membranes prior to analysis by mass spectrometry. These on-membrane
methods take approximately half the time of in-gel digestion and provide better digestion efficiency, due
to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better
protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are
commonly present.
http://www.springerlink.com/content/l227u63572311256/#section=55463&page=1
http://www.springerlink.com/content/l227u63572311256/fulltext.pdf
many biological, biochemical, and biomedical studies, and mass spectrometry has become the method of
choice for these analyses. Here we describe two methods for the on-membrane digestion of proteins electroblotted onto nitrocellulose membranes prior to analysis by mass spectrometry. These on-membrane
methods take approximately half the time of in-gel digestion and provide better digestion efficiency, due
to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better
protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are
commonly present.
http://www.springerlink.com/content/l227u63572311256/#section=55463&page=1
http://www.springerlink.com/content/l227u63572311256/fulltext.pdf
Sunday, March 18, 2012
Are amyloid-degrading enzymes viable therapeutic targets in Alzheimer’s disease? - Nalivaeva - 2011 - Journal of Neurochemistry - Wiley Online Library
Aminopeptidase A contributes to the N-terminal truncation of amyloid β-peptide - Sevalle - 2009 - Journal of Neurochemistry - Wiley Online Library
Subscribe to:
Comments (Atom)