Sample Preparation |
Protein Purification and Screening
Order
| Protocol
| Citations
Applications
- Rapid extraction of periplasmic or spheroplasmic proteins from E. coli.
- Solubilization of outer membranes in Gram-negative species.
The presence of a signal sequence is important for export of certain proteins
into the E. coli periplasmic space. The PeriPreps™
Periplasting Kit enables gentle, efficient solubilization of the outer membrane
of E. coli cells, permitting rapid extraction of proteins from the periplasmic
space. This can result in an enrichment of certain proteins compared with preparation
by lysis of the entire cell. The PeriPreps Kit combines standard periplast preparation
methods using lysozyme digestion1,2 and osmotic shock3 with
the advantages of Ready-Lyse™ Lysozyme. Ready-Lyse Lysozyme is a stable,
ready-to-use solution that is considerably more active than egg-white lysozyme.
A spheroplast lysis buffer is also provided for isolation of spheroplast proteins.
OmniCleave™ Endonuclease is included for digestion of nucleic acids, lowering
the viscosity of the sample and improving the preparation of spheroplast proteins.
The kit can also be used to extract total cellular protein.
Benefits
- Extracts periplasmic, spheroplasmic, or whole-cell proteins.
- Enriches overexpressed periplasmic proteins to as high as 95% purity in
a single step.
- Small-scale periplast preparation can be accomplished in as little as 20
minutes.
- Ready-Lyse Lysozyme is provided as a stable, ready-to-use solution.
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Figure 1. Enrichment of alkaline phosphatase in the E. coli periplasmic
fraction using the PeriPreps™ Kit. Alkaline phosphatase, a periplasmic E.
coli protein, was over-expressed from a high-copy number plasmid. One
milliliter of culture was used to prepare, in duplicate, the periplasmic
fraction (Lanes 2 and 3), the spheroplasmic fraction (Lanes 4 and 5), and
the whole cell protein (Lanes 6 and 7) using the PeriPreps Kit. Lane 1, molecular
weight marker. AP = alkaline phosphatase. BL = β-lactamase, which is
also expressed from the plasmid. |
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Figure 2. Alkaline phosphatase activity obtained using the PeriPreps™ Kit. The
average overall activities and the specific activities were determined for
the fractions described in Figure 1. One unit of alkaline phosphatase activity
causes an increase of one absorbance unit per minute when release of p-nitrophenyl
phosphate is measured at 405 nm.
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References
- Malamy, M.H. and Horecker, B.L. (1964) Biochem. 3, 1889.
- Birdsell, D.C. and Cota-Robles, E.H. (1967) J. Bact. 93, 427.
- Neu, H.C. and Heppel, L.A. (1965) J. Biol. Chem. 240, 3685.
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