Thermo Scientific Cell Lysis Solutions

lysozyme will be eliminated during subsequent washing steps in

the protocol.
DNase I: When minimizing the amount of B-PER Reagent used
in an extraction, viscosity caused by DNA may prevent efficient
B-PER Bacterial Protein Extraction Reagent centrifugation of cell debris. In such cases, add a small amount of
Yields greatly exceed those obtained using standard DNase I to clear the solution.
sonication methods. Insect cells: B-PER Reagent has been tested for the extraction of
recombinant proteins from insect cells infected by baculovirus.
Highlights: The amount of the reagent required depends on the confluency of
UÊÊ,iVœÛiÀÃÊLœÌ…Ê܏ÕLiÊ>˜`ʈ˜ÃœÕLiÊÀiVœ“Lˆ˜>˜ÌÊ«ÀœÌiˆ˜ÊvÀœ“Ê the infected cells.
bacterial lysates – purifies inclusion bodies to near-homoge- Optional/supplemental materials: Protease inhibitors, salts,
neous levels* chelating agents, reducing agents, etc. may be added directly to
UÊÊ"˜iÊi>Ãއ̜‡ÕÃiÊÀi>}i˜ÌÊqÊ«ÀœÛˆ`iÃʜ˜i‡ÃÌi«ÊE. coli cell lysis by the reagent for specific applications.
>ʓˆ`]ʘœ˜ˆœ˜ˆVÊ`iÌiÀ}i˜Ìʈ˜ÊÓäʓÊ/ÀˆÃU
ÊLÕvviÀÊ­«ÊÇ°x®
Thermo Scientific Figure 3.
UÊÊ>ÃÌÊ>˜`Êȓ«iÊqʍÕÃÌÊ>``Ê ‡* ,Ê,i>}i˜ÌÊ̜Ê>ÊL>VÌiÀˆ>Ê«iiÌÊ B-PER Reagent vs. PBS/sonication Comparison of
and shake for 10 minutes. Recover soluble proteins by pelleting Extraction Round Extraction Round Thermo Scientific
1 2 3 4 5 Pellet 1 2 3 4 5 Pellet
cell debris. Purify inclusion bodies* from the pellet using an B-PER Reagent
optimized procedure with sonication.
E. coli expressing
Uʏi݈LiÊqÊÃՈÌ>LiÊvœÀÊ>˜ÞÊÃV>iÊ«ÀœÌiˆ˜ÊiÝÌÀ>V̈œ˜ GFP was extracted
UÊÊ
ۜˆ`ÃÊVœ˜Ì>“ˆ˜>̈œ˜ÊqÊvÀiiʜvÊi˜âޓ>̈VÊVœ“«œ˜i˜ÌÃ]Ê̅ÕÃÊ five times with
avoiding contamination of the recombinant protein. If necessary, B-PER Reagent or
PBS/sonication.
the nonionic detergent can be removed by dialysis
Each extraction
UÊ
œ“«>̈LiÊ܈̅Ê-/]ÊÈ݈ÃÊ>˜`ʜ̅iÀÊ>vw˜ˆÌÞÊ«ÕÀˆwV>̈œ˜Ã1 was analyzed by
GFP**
*Does not solubilize inclusion bodies. To solubilize inclusion bodies, see page 46. SDS-PAGE.

The first step to purify or characterize a recombinant protein is to

disrupt the cell and release the protein. B-PER Bacterial Protein
Extraction Reagent offers a gentle method of bacterial cell lysis,
while also providing the most efficient way to extract recombinant **GFP = Green Fluorescent Protein
protein from E. coli. Figure 4.
300
Comparison of
B-PER Reagent Thermo Scientific
Table 1. General considerations for Thermo Scientific B-PER Reagent. PBS/sonication B-PER Reagent
250
with sonication
Three convenient reagent formats: Original B-PER Reagent is 200 for measurement
GFP Activity

ÃÕ««ˆi`ʈ˜ÊÓäʓÊ/ÀˆÃU
Ê­«ÊÇ°x®]Ê>Ì…œÕ}…Ê>Ê«…œÃ«…>ÌiÊLÕvviÀÊ of GFP. E. coli
150 expressing GFP
formulation is also available for applications requiring an amine-
free buffer. B-PER II Reagent is a twice-concentrated (2X) version was extracted five
100
times with B-PER
of the original reagent, enabling more concentrated extracts or Reagent or PBS/
50
addition to cells already in solution. sonication. Each
Fresh cells and frozen cells: B-PER Reagent is capable of extract- 0 extraction was
Pellet

1 2 3 4 5
analyzed by GFP
ing proteins from both fresh and frozen cells. However, the extrac- Rounds of Extraction activity assay.
tion is typically most effective with frozen cells.
E. coli strains: B-PER Reagents work well for many common bac- Reference
1. Dorsey, C.W., et al. (2003). Genetic organization of an Acinetobacter baumannii
terial host strains. They are especially suitable for the commonly chromosomal region harbouring genes related to siderophore biosynthesis and transport.
used, protease-defective bacterial expression host BL21 strains. If Microbiology 149, 1227-1238.
the lysis is not efficient for a particular bacterial strain, try freezing
the cells before extraction. Ordering Information
Soluble proteins and inclusion bodies: Perform a mini-scale
extraction to determine solubility of recombinant proteins before Product # Description Pkg. Size
performing larger-scale extractions and purifications. Recombinant 78248 B-PER Bacterial Protein Extraction Reagent 200 ml
proteins expressed in bacteria often form inclusion bodies, espe-
78243 B-PER Bacterial Protein Extraction Reagent 165 ml
cially when expressed at high levels. B-PER Reagents effectively
extract and remove soluble proteins from the insoluble inclusion 89833 Lysozyme 5g
body pellet. When combined with inclusion body solubilization and 89835 DNase I 5,000 units
protein refolding, purified recombinant protein may be obtained
78425 Halt Protease Inhibitor Single-Use Cocktail, 24 x 100 μl
from the inclusion body pellet. EDTA-free (100X) microtubes
Lysozyme: For inclusion body purification, add lysozyme to digest
78430 Halt Protease Inhibitor Single-Use Cocktail 24 x 100 μl
cell debris and release the inclusion bodies. Lysozyme digestion (100X) microtubes
can significantly improve inclusion body protein purity; the Includes 0.5 M EDTA Solution (100X), 2.5 ml

8 For more information, or to download product instructions, visit www.thermo.com/pierce