Sample Preparation |
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Applications
- Lysis of Gram-negative or Gram-positive bacteria (Table 1) for protein purification
(Fig. 1).
- Purification of nucleic acids from bacteria.
Ready-Lyse™ Lysozyme Solution is a recombinant lysozyme preparation,
derived from a nonmammalian, nonavian source, that is useful for the lysis of
Gram-negative (such as E. coli) and Gram-positive (such as Bacillus sp.)
bacteria. The specific activity of Ready-Lyse Lysozyme is 200-fold higher than
the specific activity of egg-white lysozyme and, therefore, less lysozyme is
needed in a reaction. Also, unlike egg-white lysozyme, Ready-Lyse Lysozyme Solution
is stable at -20°C, eliminating the need to prepare a fresh solution for
each use. The use of Ready-Lyse Lysozyme results in higher yields of protein
than can be obtained with standard eggwhite lysozyme.
Benefits
- No sample agitation or heat generation necessary that can result in protein
denaturation.
- Volumes easily adjusted for preparative yields.
Unit Definition: One unit of Ready-Lyse Lysozyme produces a decrease
in A350 of 0.001 per minute at 23°C with a 0.5 mg/ml suspension
of lyophilized E. coli K802 cells in 50 mM Tris-HCl (pH 7.5).
Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5),
0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100. |
Escherichia coli
Salmonella typhimurium
Actinobacillus pleuropneumoniae
Rhodobacter sphaeroides
Shewanella putrefaciens
Flavobacteria odoratum |
Oerskovia xanthinolytica
Bacillus subtilis |
Table 1. Bacteria lysed with Ready-Lyse™ Lysozyme Solution. |
 |
Figure 1. Lysis with Ready-Lyse™ Lysozyme increases
yields of nucleic acids. 500 µg per ml of pHC79 cosmid DNA was
incubated for 15 minutes at 22°C in TE buffer (25 mM Tris (pH 8.0), 10
mM EDTA) containing either 5 µg (30 KU) per ml of Ready-Lyse Lysozyme
or 500 µg (25 KU) per ml of egg white lysozyme. The solutions were
centrifuged for 10 minutes and the pellets were resuspended in TE buffer
containing 0.1% SDS. DNA in supernatants and pellets was separated by electrophoresis
in a 0.8% agarose gel. Approximately 50% of the DNA was lost due to precipitation
by egg white lysozyme (EW), while Ready-Lyse Lysozyme (RL) caused minimal
precipitation losses of DNA compared to control (C) samples without lysozyme. |
 |
Figure 2. Use of Ready-Lyse™ Lysozyme Solution to
recover recombinant proteins. One ml of induced cells from a recombinant E.
coli clone was pelleted by microcentrifugation before induction and at
1 and 3 hours after induction. Each sample was resuspended in 50 µl
of cold TEBG buffer. One µl of Ready-Lyse Solution was added to each
suspension and the cells were incubated at room temperature for 30 minutes.
The cell debris was pelleted and 10 µl of the supernatant were run
on an SDS-PAGE gel. Lane 1, molecular weight markers; Lanes 2-4, time points
of induction. The induced protein is designated by an arrow. |
MasterPure™ Gram Positive DNA Purification Kit
PeriPreps™ Periplasting Kit (for periplasmic proteins)
ReadyPreps™ Protein Preparation Kit (for total cellular proteins)
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