Cell Lysis Protocol
•
Add lysozyme and DNase I to Lysis reagent for the most efficient extraction; however, the extraction of some overexpressed proteins does not require the addition of lysozyme. If the addition of lysozyme and DNase I might interfere
with the downstream application, the use of these enzymes should be omitted.
•
Some over-expressed proteins can be insoluble, misfolded or expressed in inclusion bodies. The best way to obtain
soluble protein is to adjust the expression conditions; however, if this is not possible, the Thermo Scientific Inclusion
Body Solubilization Reagent (Product No. 78115) can be used.
•
Whole cell lysates prepared with the Lysis Reagent are compatible with the Thermo Scientific Pierce BCA Protein Assay
Kit.
Procedure for Extracting Protein from Bacteria (Extraction to be done on both negative
control and positive control) (pGLO positive pellet : pGLO negative pellet) (5 Pellets :
5 Pellets).
WorkingLysis Buffer = Lysis Buffer + DNAse + Lysozyme + PIC (see volumes below)
1.
Calculate the amount of total lysis buffer needed to make Working Lysis Buffer
2.
Remove pelleted bacterial cells from -20 C, and thaw on ice.
3.
Add 2 µL of lysozyme and 2 µL of DNase I per 1mL lysis reagent. Add 1 ul EDTA- free protease inhibitors (PIC).
4.
Add 350 ul of Lysis Reagentworking reagent to cell pellet. Pipette the suspension up and down until it is homogeneous.
5.
Incubate 30 minutes in a rack in -20 C, followed by 20 minutes on water bath at 37 C, followed by 10 minutes on ice.
6.
Centrifuge the lysates @ 10,000 x g for 5 minutes and collect the supernatant transfer to new microtubes, you should
have 8 microtubes (6 pGLO positive tubes and 2 pGLO negative tubes)
7.
Sonicate samples for 10 secs.
8.
Begin the BCA assay protocol to determine protein concentration of all unknowns generated (8 analytes that were
generated). (See Protocol from Week 2).
Troubleshooting
Problem
Protein of interest is not
solubilized
Possible Cause
Protein of interest is expressed
in inclusion bodies
Bacteria are not lysed upon
enzyme addition
Temperature is too low
Viscosity of extract is high
Excessive over-expressed
protein remains in the pellet
B-PER Reagent is cloudy after
shipping
Lysozyme has low activity or is
inactive
DNase I has low activity or is
inactive
Protein is too large for B-PER
Reagent alone
Reagent is cold
Solution
Adjust expression conditions or use an inclusion
body solubilization reagent
Add lysozyme and DNase I to the B-PER Reagent
Warm culture and other reagents to room
temperature
Add more lysozyme to the B-PER Reagent or
purchase new enzyme
Add more DNase I to the B-PER Reagent or
purchase new enzyme
Add Mg2+ ions to 2mM final concentration
Add lysozyme and DNase I to the B-PER Reagent
Warm reagent to room temperature
Pierce Biotechnology
PO Box 117
(815) 968-0747
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
2
www.thermoscientific.com/pierce