|
|
LYSIS BUFFER |
COMPATIBLE
REPORTER ASSAY |
PROTEIN ASSAY (4) |
WESTERN BLOT (5) |
|
|
Cell Culture Lysis Reagent (CCLR) |
- Luciferase
Assay System (1)
- β-galactosidase assay (β-Galactosidase
Enzyme Assay System with Reporter Lysis Buffer; 2)
|
- GenoTechnology NI™ Protein Assay
- Pierce BCA Protein Assay with Compat-Able™ Protein Assay Preparation
Reagent Set
- Bio-Rad RC DC™ Protein Assay
- Molecular Probes NanoOrange® Protein Quanitation Kit
(dilute lysate 1:10)
- Bio-Rad DC Protein Assay or Pierce BCA Protein Assay (dilute lysate
at least 1:2)
|
- Compatible
- Can release cytoplasmic, mitochondrial and nuclear contents (6)
|
|
|
LYSIS BUFFER |
COMPATIBLE
REPORTER ASSAY |
PROTEIN ASSAY (4) |
WESTERN BLOT (5) |
|
|
Reporter Lysis Buffer (RLB) |
- Luciferase
Assay System (1)
- β-galactosidase assay (includes both
β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer
and Beta-Glo®
Assay System)
- CAT Enzyme
Assay System with Reporter Lysis Buffer
|
- GenoTechnology NI™ Protein Assay
- Pierce BCA Protein Assay with Compat-Able™ Protein Assay Preparation
Reagent Set
- Pierce Coomassie® Plus protein assay
- Bio-Rad RC DC™ Protein Assay
- Normal Bradford assay with 2–8µl of undiluted lysate
- Molecular Probes NanoOrange® Protein Quanitation Kit
(dilute lysate 1:10)
- Bio-Rad DC Protein Assay (large scale only) or Pierce BCA Protein
Assay (dilute lysate at least 1:2)
|
- Compatible
- Can release cytoplasmic and nuclear contents. RLB releases nuclear
contents more efficiently with a freeze-thaw cycle (6)
|
|
|
LYSIS BUFFER |
COMPATIBLE
REPORTER ASSAY |
PROTEIN ASSAY (4) |
WESTERN BLOT (5) |
|
|
Passive Lysis Buffer (PLB) |
- Luciferase
Assay System (1)
- β-galactosidase assay (includes both
β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer
and Beta-Glo®
Assay System)
- CAT Enzyme
Assay System with Reporter Lysis Buffer
- Dual-Luciferase®
Reporter Assay System
|
- GenoTechnology NI™ Protein Assay
- Pierce BCA Protein Assay with Compat-Able™ Protein Assay Preparation
Reagent Set
- Pierce Coomassie® Plus protein assay
- Bio-Rad RC DC™ Protein Assay
- Pierce BCA Protein Assay (dilute lysate 1:10)
- Molecular Probes NanoOrange® Protein Quanitation Kit
(dilute lysate 1:10)
|
- Compatible
- Can release cytoplasmic and nuclear contents (6)
|
|
|
LYSIS BUFFER |
COMPATIBLE
REPORTER ASSAY |
PROTEIN ASSAY (4) |
WESTERN BLOT (5) |
|
|
Glo Lysis Buffer (GLB) |
- Luciferase
Assay System (1)
- Bright-Glo™
Luciferase Assay and
Steady-Glo® Luciferase Assay System
- ONE-Glo™
Luciferase Assay System
- β-galactosidase assay (includes both
β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer
and Beta-Glo®
Assay System; up to 45µl of lysate in assay; 2)
- CAT Enzyme
Assay System with Reporter Lysis Buffer (up to 25µl of lysate in
assay; 3)
|
- GenoTechnology NI™ Protein Assay
- Pierce BCA Protein Assay with Compat-Able™ Protein Assay Preparation
Reagent Set
- Bio-Rad RC DC™ Protein Assay
- Pierce BCA Protein Assay (dilute lysate 1:10)
- Molecular Probes NanoOrange® Protein Quanitation Kit
(dilute lysate 1:10)
|
- Compatible
- Can release cytoplasmic, mitochondrial and nuclear contents (6)
|
|
|
LYSIS BUFFER |
COMPATIBLE
REPORTER ASSAY |
PROTEIN ASSAY (4) |
WESTERN BLOT (5) |
|
|
Renilla Luciferase Assay Lysis Buffer (RLALB) |
- Renilla
Luciferase Assay System
- β-galactosidase assay (up to 45µl of lysate in assay)
- CAT Enzyme
Assay System with Reporter Lysis Buffer (up to 25µl of lysate in
assay)
|
- GenoTechnology NI™ Protein Assay
- Pierce BCA Protein Assay with Compat-Able™ Protein Assay Preparation
Reagent Set
- Bio-Rad RC DC™ Protein Assay
- Molecular Probes NanoOrange® Protein Quanitation Kit
(dilute lysate 1:10)
|
|
|
(1)Luciferase assays include all Promega assays that are designed to measure
only the activity of firefly luciferase and do not have extended signal
half-life. These include products with catalog numbers E1500, E1501, E4030,
E4530, E4550 and E1483.
(2)May be used in place of 1X RLB to lyse cells and dilute cell lysates in
the β-Galactosidase Enzyme Assay, provided that a different stop buffer is
used. We recommend the use of 1M Tris base as a stop buffer when samples are
in 1X CCLR and 1X GLB because addition of the 1M Sodium Carbonate (provided
with the system) causes a precipitate to form and absorbance readings are no
longer accurate.
(3)Activity will not be as high as that detected in RLB. If CAT
expression is very low, RLB may be necessary for optimal sensitivity.
(4)When performing a protein assay using any of the suggested protein
assay systems, an equal concentration of lysis buffer must be used to
generate the standard curve as well for optimal accuracy. If problems arise
with the protein assay, further dilute the lysate with water and perform the
protein assay on the macro, not micro, scale.
(5)Western blot analysis was performed using either AP- or HRP-conjugated
secondary antibodies and colorimetric detection with TMB stabilized
substrates for HRP or Western Blue® for AP. 1X, 2X and 5X
concentrations of the Promega lysis buffers were tested.
(6)The ability of the lysis reagents to lyse different cellular organelles
(cell membrane, nucleus, mitochondria) was investigated. This was done by
probing the Western blots for a cytoplasmic antigen (ERK1/2), a
mitochondrial antigen (cytochrome oxidase subunit 1), or a nuclear antigen
(histone). The efficiency of lysis may depend on growth conditions and cell
or tissue type.
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