Mouse liver tissue samples were processed with three different sample
preparation kits to extract and enrich specific subclasses of proteins.
Instructions for each kit were followed exactly. Protease inhibitors were
added to the starting buffers immediately prior to use to prevent proteolysis
during extraction. Following extraction, if required, protein samples
were processed with the ReadyPrep 2-D cleanup kit to remove salts and
detergents and to create a low-conductivity sample suitable for isoelectric
focusing (IEF). This cleanup kit was also employed, if required, to concentrate
proteins from dilute samples. Prior to IEF all samples were solubilized
in PSB, to which 50 mM DTT, 2 mM TBP, and 0.2% Bio-Lyte
ampholytes (310)
were added, and applied to 17 cm, pH 310 NL, ReadyStrip IPG strips.
Approximately 455 g of protein was loaded to each strip. E. coli samples
were processed with the ReadyPrep membrane II kit protocol. Extracted
membrane proteins were applied to 24 cm, pH 47 ReadyStrip IPG strips.
2-D Electrophoresis
The IPG strips were focused using a PROTEAN IEF cell for ~60,000 V-hr
at a final focusing voltage of 10,000 V. The cell was set for rapid voltage
ramping. Focused IPG strips were loaded onto 816% SDS-PAGE gels following
equilibration for 10 min with DTT containing buffer followed by 10 min
with iodoacetamide containing buffer. Following electrophoresis, gels
were fixed for 30 min, stained with Bio-Safe Coomassie stain for 1 hr,
and then destained for at least 2 hr before scanning with a GS-800 densitometer.
Protein Identification
2-D gel images were processed for analysis with PDQuest 2-D analysis
soft
'"/>
Source:
Page: All 1 2 3 4 5 6 Related biology technology :1.
Sample Preparation for Microinjection2.
GeneChip Sample Cleanup Module for cleanup of cDNA and in vitro
transcribed cRNA3.
Quantitation of the Protein MIA as a Marker for
Chondrocytes in Research Samples4.
A Better Way to Isolate RNA AND Protein From the Same Sample5.
Isolate Total RNA and Protein From the Same Sample6.
Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis7.
Preserve Samples for RNA Expression Microarrays RNAlater Around the World8.
RNA from LCM Samples9.
RNA, DNA, and Protein from a Single Sample10.
Robust High Throughput RNA Isolation from Blood Samples11.
RNAlater Around the World: The Art of Processing Difficult Samples