Proteasome-Glo检测试剂盒说明书

Technical Bulletin

Proteasome-Glo™

Assay Systems

INSTRUCTIONS FOR USE OF PRODUCTS G8531, G8532, G8621, G8622,

G8631, G8632, G8641, ANDG8642.

PRINTED IN USA.

Revised 5/09Part# TB349

Proteasome-Glo™ Assay Systems

All technical literature is available on the Internet at: /tbs/

Please visit the web site to verify that you are using the most current version of this

Technical Bulletin. Please contact Promega Technical Services if you have questions on use

of this system. E-mail: techserv@

1

t Components and .7

10

for Detection of 11

11

rd Assay (96-well, 100µl Final Reaction Volume).............................12

13

nces ........................................................................................................14

d Products .............................................................................................15

ption

The Proteasome-Glo™ 3-Substrate System

(a,b,c)

consists of three homogeneous

bioluminescent assays that measure the three proteolytic activities associated

with the proteasome (each of these three assays is also available separately).

The proteasome is a multicatalytic complex in the nucleus and cytosol of all

eukaryotic cells that is responsible for proteolysis of ubiquitin-tagged proteins.

The catalytic core of the complex, the 20S proteasome, is a barrel-shaped

assembly of 28 protein subunits that possesses three different proteolytic

activities designated as chymotrypsin-like, trypsin-like, and caspase-like (also

termed post-glutamyl peptide hydrolase; 1,2). The catalytic sites are located on

the inner surface of the central β-rings of the cylindrical particle, and access to

them is controlled by narrow gated channels in the outer α-rings of the

complex. The association of the 20S particle with a 19S regulatory complex at

one or both ends of the barrel forms the 26S proteasome and confers an open-

channel conformation, resulting in much higher rates of peptide hydrolysis

(3,4). The 26S proteasome degrades polyubiquitinated proteins in an ATP-

dependent manner. The 19S regulatory unit binds and removes the ubiquitin

chains from tagged proteins, and ATPases within the regulatory complex

appear to unfold protein substrates and translocate the unfolded polypeptides

into the 20S core (2–5). The 20S catalytic core and the 19S regulatory complex

are highly conserved from yeast to mammals (1). The 26S proteasome complex

processes aberrant and misfolded proteins as well as proteins regulating cell

cycle, growth and apoptosis and is essential for cellular function.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Printed in USA.

Revised 5/09

Part# TB349

Page 1

The role of the proteasome in degrading several important regulatory proteins

has led to the identification of the proteasome as a therapeutic target for cancer

treatment. Proteasome inhibitors can induce apoptosis, and interestingly,

transformed cells display greater susceptibility to proteasome inhibition than

nonmalignant cells (5). The enhanced proliferative rate of malignant cells may

cause accumulation of damaged proteins at a higher rate, which in turn may

increase dependency on proteasomal degradation (6). The first-generation

proteasome inhibitor, bortezomib (PS-341), is now an approved drug for the

treatment of refractory multiple myeloma, and second-generation inhibitors are

currently being developed (7).

The Proteasome-Glo™ 3-Substrate System provides three separate assays that

differ in their ability to detect different protease activities based on their

substrate components. The luminogenic substrates provided for the

chymotrypsin-like, trypsin-like, and caspase-like activities are Suc-LLVY-

aminoluciferin, Z-LRR-aminoluciferin, and Z-nLPnLD-aminoluciferin,

respectively. Each substrate is added to a buffer system optimized for

proteasome activity and luciferase activity to make a Proteasome-Glo™

Reagent for a particular catalytic activity. The individual Proteasome-Glo™

Reagent is added to test samples in an “add-mix-measure” format, resulting in

proteasome-induced cleavage of the particular substrate. Substrate cleavage

generates a “glow-type” luminescent signal produced by the luciferase reaction

(Figure 1). In this homogeneous coupled-enzyme format, the signal is

proportional to the amount of proteasome activity (Figure 2).

The Proteasome-Glo™ Reagents rely on the properties of a proprietary

thermostable luciferase (Ultra-Glo™ Recombinant Luciferase

(b)

) that is

formulated to generate a “glow-type” luminescent signal and provides

excellent performance across a wide range of assay conditions. The proteasome

and luciferase enzyme activities reach a steady-state such that the luminescent

signal peaks rapidly and is maintained for several hours with minimal loss of

signal (Figure 3). The Proteasome-Glo™ Assays provide rapid, sensitive, and

accurate assays for the three proteolytic activities of the proteasome (Figures 4

and 5). The homogeneous Proteasome-Glo™ Assays are designed for use with

multiwell-plate formats, making them ideal for automated high-throughput

screening of proteasome activity and inhibition (Figure 6).

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Part# TB349

Page 2

Printed in USA.

Revised 5/09

H

Suc-LLVY – N

or

Z-LRR –

or

Z-nLPnLD –

S

N

N

S

COOH

Proteasome

Suc-LLVY +

H

2

N COOH S N

or

Z-LRR

N S

or

Aminoluciferin

Z-nLPnLD

Ultra-Glo™ rLuciferase

ATP, Mg

++

, O

2

Figure 1. The luminogenic substrates containing the Suc-LLVY, Z-LRR, or Z-

nLPnLD sequence are recognized by the 20S proteasome. Following cleavage by

the 20S proteasome, the substrate for luciferase (aminoluciferin) is released, allowing

the luciferase reaction to produce light.

Suc-LLVY-aminoluciferin

y = 1.0394x + 5.4385

r

2

= 0.993

y = 0.9922x + 4.7521

r

2

= 0.998

10,000,000

Z-LRR-aminoluciferin

Z-nLPnLD-aminoluciferin

L

u

m

i

n

e

s

c

e

n

c

e

(

R

L

U

,

b

a

c

k

g

r

o

u

n

d

-

s

u

b

t

r

a

c

t

e

d

)

1,000,000

100,000

10,000

1,000

100

10

1

0

1

0

0

y = 1.0593x + 5.927

r

2

= 0.998

20S (µg/ml)

Figure 2. Luminescence is proportional to proteasome concentration. Titrations of

20S proteasome were performed in 96-well plates using the Proteasome-Glo™

3-Substrate System. Human

20S proteasome was serially diluted in 10mM

HEPES (pH 7.6). Thirty minutes after adding the individual Proteasome-Glo™

Reagents separately, luminescence was recorded as relative light units (RLU)

on a GloMax

®

96 Microplate Luminometer. The results were linear over 4 logs

of 20S proteasome concentration for all three point represents the

average of four wells. The background (blank without 20S) was subtracted from

each. Values for r

2

and slope were calculated after transforming the data to a

log

10

-log

10

plot.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Printed in USA.

Revised 5/09

Part# TB349

Page 3

5

8

6

2

M

A

0

.

0

0

0

.

0

0

1

0

.

0

0

1

0

.

0

1

0

.

1

1

.

0

1

0

5

8

5

5

M

A

Light

A.

Suc-LLVY-Glo™ Substrate + 20S

Z-LRR-Glo™ Substrate + 20S

Z-nLPnLD-Glo™ Substrate + 20S

Suc-LLVY-Glo™ Substrate, no 20S

Z-LRR-Glo™ Substrate, no 20S

Z-nLPnLD-Glo™ Substrate, no 20S

1,000,000

L

u

m

i

n

e

s

c

e

n

c

e

(

R

L

U

)

100,000

10,000

1,000

100

Time (minutes)

B.

Suc-LLVY-Glo™ Substrate + 20S

Z-LRR-Glo™ Substrate + 20S

300,000

250,000

200,000

150,000

100,000

50,000

0

5

8

6

3

M

A

L

u

m

i

n

e

s

c

e

n

c

e

(

R

L

U

)

040

80120160

Time (minutes)

Figure 3. Signal stability of the Proteasome-Glo™ Assay Systems. Human purified

20S proteasome (1µg/ml) was assayed in 96-well plates using the individual

Proteasome-Glo™ Assays. Luminescence was monitored at various time points

over 3 hours on a GloMax

®

96 Microplate Luminometer. Panel signals peak

rapidly and then are very stable for all three assays as shown on a log scale.

Panel stable signals generated with 20S proteasome are shown for the

chymotrypsin-like (Suc-LLVY-Glo™ ) and trypsin-like (Z-LRR-Glo™) Substrates on

a linear scale.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Part# TB349

Page 4

Printed in USA.

Revised 5/09

A.

Suc-LLVY-Glo™ Substrate

Suc-LLVY-AMC

Suc-LLVY-AMC + SDS

100,000

S

i

g

n

a

l

-

t

o

-

N

o

i

s

e

R

a

t

i

o

10,000

1,000

100

10

1

0.1

0

0

.

0

0

0

1

1

1

0

0

0

.

0

0

0

.

0

0

.

0

1

0

.

1

1

.

0

1

0

20S (µg/ml)

Boc-LRR-AMC

Ac-nLPnLD-AMC

B.

Z-LRR-Glo™ Substrate

Z-nLPnLD-Glo™ Substrate

100,000

S

i

g

n

a

l

-

t

o

-

N

o

i

s

e

R

a

t

i

o

10,000

1,000

100

10

1

20S (µg/ml)

Figure 4. Sensitivity of the Proteasome-Glo™ Assays compared to fluorescent

assays. Human purified 20S proteasome was titrated and assayed in 96-well plates

using the Proteasome-Glo™ 3-Substrate System or comparable fluorogenic

substrates. Luminescence and fluorescence were monitored at 30 minutes on a

GloMax

®

96 Microplate Luminometer or a Labsystems Fluoroskan Ascent plate

reader, respectively. The results are plotted as signal-to-noise ratios. The limit of

detection is defined as the amount of 20S proteasome giving a signal-to-noise

ratio >3 (dashed lines). Panel Proteasome-Glo™ Chymotrypsin-Like Assay

(Suc-LLVY-Glo™ Substrate) was compared to a Suc-LLVY-AMC substrate. The

fluorescent assay was performed with or without 0.02% SDS. Panel

Proteasome-Glo™ Trypsin-Like Assay (Z-LRR-Glo™ Substrate) was compared to

the fluorogenic substrate, Boc-LRR-AMC, and the Proteasome-Glo™ Caspase-Like

Assay (Z-nLPnLD-Glo™ Substrate) was compared to the fluorogenic substrate, Ac-

nLPnLD-AMC. The signal-to-noise ratios are greater, and the limits of detection are

significantly lower for all of the luminescent proteasome assays compared to the

fluorescent proteasome assays.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Printed in USA.

Revised 5/09

Part# TB349

Page 5

5

8

6

4

M

A

0

0

.

0

0

0

1

0

0

.

0

0

1

0

.

0

0

1

1

0

.

0

0

.

1

1

.

0

1

0

160,000

L

u

m

i

n

e

s

c

e

n

c

e

(

R

L

U

)

140,000

120,000

100,000

80,000

60,000

40,000

20,000

0

0.0010.010.11

Clasto-lactacystin β-lactone (µM)

Figure 5. Determination of IC

50

values. The inhibitor concentration that results in

50% inhibition (IC

50

) was determined for the proteasome irreversible inhibitor,

clasto-lactacystin β-lactone (8), using the Proteasome-Glo™ Chymotrypsin-Like

Assay. The inhibitor was resuspended in DMSO, serially diluted in 10mM HEPES

(pH 7.6) and combined with 1µg/ml 26S proteasome (Biomol) in 96-well plates.

Reagent containing the Suc-LLVY-Glo™ Substrate was added after one hour, and

luminescence was recorded 10 minutes after reagent addition. GraphPad Prism

®

software was used to calculate the IC

50

, which was 30nM.

Assay Advantages

Broad Dynamic Range:The assays are linear over 4 logs of proteasome

concentrations and can detect 20S proteasome at concentrations as low as

0.5ng/ml (1pM) (Figures 2 and 4).

Fast:Maximum sensitivity is reached in 10–30 minutes after adding reagent

(Figure 3), because the assays are not dependent on accumulation of cleaved

product for sensitivity.

Greater Sensitivity:The coupled-enzyme format and the speed of the

Proteasome-Glo™ Assay results in low background and excellent signal-to-

noise ratios. The assays are significantly more sensitive than fluorescence-based

proteasome assays (Figure 4).

Accurate: The broad linear range and excellent sensitivity readily translate to

accurate kinetic analysis of inhibitors (Figure 5).

Simplified Method: The “add-mix-read” protocols make the assays amenable

to automation (Figure 6).

Amenable to Batch Processing:The coupled-enzyme, homogeneous format

results in a stable, glow-type signal, allowing flexibility in read time once the

reagent is added (Figure 3).

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Part# TB349

Page 6

Printed in USA.

Revised 5/09

5

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t Components and Storage Conditions

ProductSizeCat.#

Proteasome-Glo™ 3-Substrate System10mlG8531

Includes 3 individual kits. Each kit contains sufficient reagent for 100 assays at

100µl/assay or 200 assays at 50µl/assay in 96-well plates or 400 assays at 25µl/assay in

384-well plates. Includes:

•

•

•

1 kit

1 kit

1 kit

Proteasome-Glo™ Chymotrypsin-Like Assay (Cat.# G8621)

Proteasome-Glo™ Trypsin-Like Assay (Cat.# G8631)

Proteasome-Glo™ Caspase-Like Assay (Cat.# G8641)

Cat.#

G8532

ProductSize

Proteasome-Glo™ 3-Substrate System50ml

Includes 3 individual kits. Each kit contains sufficient reagent for 500 assays at

100µl/assay or 1,000 assays at 50µl/assay in 96-well plates or 2,000 assays at

25µl/assay in 384-well plates. Includes:

•

•

•

1 kit

1 kit

1 kit

Proteasome-Glo™ Chymotrypsin-Like Assay (Cat.# G8622)

Proteasome-Glo™ Trypsin-Like Assay (Cat.# G8632)

Proteasome-Glo™ Caspase-Like Assay (Cat.# G8642)

Items Available Separately

Product

Proteasome-Glo™ Chymotrypsin-Like Assay

Includes:

•

•

•

10ml

1 bottle

50µl

Proteasome-Glo™ Buffer

Luciferin Detection Reagent

Suc-LLVY-Glo™ Substrate

Size

50ml

Cat.#

G8622

Size

10ml

Cat.#

G8621

Product

Proteasome-Glo™ Chymotrypsin-Like Assay

Includes:

•

•

•

50ml

1 bottle

250µl

Proteasome-Glo™ Buffer

Luciferin Detection Reagent

Suc-LLVY-Glo™ Substrate

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Printed in USA.

Revised 5/09

Part# TB349

Page 7

t Components and Storage Conditions (continued)

Size

10ml

Cat.#

G8631

Product

Proteasome-Glo™ Trypsin-Like Assay

Includes:

•

•

•

10ml

1 bottle

100µl

Proteasome-Glo™ Buffer

Luciferin Detection Reagent

Z-LRR-Glo™ Substrate

Product

Proteasome-Glo™ Trypsin-Like Assay

Includes:

•

•

•

50ml

1 bottle

500µl

Proteasome-Glo™ Buffer

Luciferin Detection Reagent

Z-LRR-Glo™ Substrate

Size

50ml

Cat.#

G8632

Product

Proteasome-Glo™ Caspase-Like Assay

Includes:

•

•

•

10ml

1 bottle

50µl

Proteasome-Glo™ Buffer

Luciferin Detection Reagent

Z-nLPnLD-Glo™ Substrate

Size

10ml

Cat.#

G8641

Product

Proteasome-Glo™ Caspase-Like Assay

Includes:

•

•

•

50ml

1 bottle

250µl

Proteasome-Glo™ Buffer

Luciferin Detection Reagent

Z-nLPnLD-Glo™ Substrate

Size

50ml

Cat.#

G8642

Storage Conditions: Store the Proteasome-Glo™ Assays at –20°C protected

from light. The Proteasome-Glo™ Buffer may be thawed and stored at 4°C for 2

months with no loss in signal. The Proteasome-Glo™ Substrates may be

refrozen and stored at –20°C with minimal loss of signal. Proteasome-Glo™

Reagent (combined Proteasome-Glo™ Substrate, Proteasome-Glo™ Buffer, and

Luciferin Detection Reagent) can be stored at 4°C or –20°C for 1 month with

minimal loss of activity.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Part# TB349

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Printed in USA.

Revised 5/09

Proteasome-Glo™

Buffer

Luciferin

Detection

Reagent

Substrate

Proteasome-Glo™

Reagent

Add the substrate and

incubate at room

temperature for

60 minutes.

Label the reagent

to indicate the

substrate used.

Add equal volume

of Proteasome-Glo™

Reagent to samples.

Mix. Incubate 10

minutes to 3 hours.

Measure

luminescence.

5

8

5

6

M

A

Figure 6. Flow diagram showing preparation and use of the

Proteasome-Glo™ Reagent.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Printed in USA.

Revised 5/09

Part# TB349

Page 9

t Preparation

Directions are given for performing the Proteasome-Glo™ Assays in a total

volume of 100µl using 96-well plates and a luminometer. However, the assays

can be easily adapted to different volumes if a 1:1 ratio of Proteasome Glo™

Reagent volume to sample volume is preserved (e.g., 25µl of sample + 25µl

Proteasome-Glo™ Reagent in a 384-well format).

Materials to be Supplied by the User

•

•

•

•

•

•

white-walled multiwell plates (black plates may be used, but RLU will be

reduced)

multichannel pipette or automated pipetting station

plate shaker

luminometer capable of reading multiwell plates

20S proteasome enzyme (e.g., Biomol Cat.# PW8720 or Boston Biochem

Cat.# E-360)

10mM HEPES Buffer (pH 7.6, for proteasome dilution)

Proteasome-Glo™ Reagent Preparation

1.

2.

Thaw the Proteasome-Glo™ Buffer and equilibrate both buffer and the

lyophilized Luciferin Detection Reagent to room temperature before use.

Reconstitute the Luciferin Detection Reagent in the amber bottle by adding the

appropriate volume of Proteasome-Glo™ Buffer (10ml for Cat.# G8621, G8631,

G8641; 50ml for Cat.# G8622, G8632, G8642). The Luciferin Detection Reagent

should go into solution easily in less than one minute.

Thaw the appropriate substrate and equilibrate to room temperature before

use. For the Chymotrypsin-Like Assay, use the Suc-LLVY-Glo™ Substrate; for

the Trypsin-Like Assay, use the Z-LRR-Glo™ Substrate; and for the Caspase-

Like Assay, use the Z-nLPnLD-Glo™ Substrate. A slight precipitate may be

observed. Mix well by vortexing briefly.

Prepare the Proteasome-Glo™ Reagent by adding the Proteasome-Glo™

Substrate to the resuspended Luciferin Detection Reagent as per Table 1.

Label the reagent bottle to identify the substrate used.

Allow the Proteasome-Glo™ Reagent to sit at room temperature for 60

minutes before use. This allows the removal of any contaminating free

aminoluciferin. Although free aminoluciferin is not detected by HPLC, it is

present in trace amounts (Figure 7).

Note: The Protoeasome-Glo™ Reagent (combined Proteasome-Glo™

Substrate, Proteasome-Glo™ Buffer and Luciferin Detection Reagent) can be

stored at 4°C or –20°C for 1 month with minimal loss of activity.

3.

4.

5.

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Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Part# TB349

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Printed in USA.

Revised 5/09

Table 1. Volume of Substrate Added to Create Proteasome-Glo™ Reagent.

Volume of

Substrate

Added

50µl

250µl

100µl

500µl

50µl

250µl

Substrate

Concentration

in Reagent

40µM

40µM

30µM

30µM

40µM

40µM

Proteasome-Glo™ Assay

Chymotrypsin-Like Assay

Chymotrypsin-Like Assay

Trypsin-Like Assay

Trypsin-Like Assay

Caspase-Like Assay

Caspase-Like Assay

Cat.#Substrate

G8621Suc-LLVY-Glo™

G8622Suc-LLVY-Glo™

G8631Z-LRR-Glo™

G8632Z-LRR-Glo™

G8641Z-nLPnLD-Glo™

G8642Z-nLPnLD-Glo™

10,000,000

L

u

m

i

n

e

s

c

e

n

c

e

(

R

L

U

)

1,000,000

100,000

10,000

1,000

100

0

25

Suc-LLVY-Glo™ Substrate

Z-LRR-Glo™ Substrate

Z-nLPnLD-Glo™ Substrate

Time (minutes)

Figure 7. Time course of free aminoluciferin removal from the Proteasome-Glo™

Reagents. The proteasome substrates (Suc-LLVY-Glo™, Z-LRR-Glo™, and

Z-nLPnLD-Glo™ Substrates) were added to the individual bottles of reconstituted

Luciferin Detection Reagent, and a time course of luminescence loss was recorded.

Trace amounts of free aminoluciferin are present in the substrate and are removed

by incubation with the reconstituted Luciferin Detection Reagent. To achieve

maximal assay sensitivity with minimal background luminescence, the prepared

Proteasome-Glo™ Reagent should be incubated for 60 minutes at room temperature

before use.

for Detection of Proteasome Activity

Prepare the following reactions to detect proteasome activity (or inhibition of

activity) in purified enzyme preparations:

•Blank:Proteasome-Glo™ Reagent + vehicle control for test compound or

inhibitor, if used.

Conditions

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Printed in USA.

Revised 5/09

Part# TB349

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75

125

175

225

•Positive Control: Proteasome-Glo™ Reagent + vehicle control + purified

proteasome enzyme (20S or 26S).

•Assay:Proteasome-Glo™ Reagent + test compound + purified proteasome

enzyme (20S or 26S).

The blank is used as a measure of any background luminescence associated

with the test compound vehicle and Proteasome-Glo™ Reagent and should be

subtracted from experimental values. The positive control is used to determine

the maximum luminescence obtainable with the purified enzyme system.

Vehicle refers to the solvent used to dissolve the inhibitor or test compound

used in the study.

Notes:

e the Proteasome-Glo™ Reagent as described in Section

3, and mix

thoroughly before starting the assay.

final concentration of proteasome should be within the linear range of

the assay (Figure 2).

recommended proteasome dilution buffer is 10mM HEPES (pH 7.6).

identical enzyme concentrations for the assay and positive control

reactions.

gentle mixing you may use a plate shaker.

maximal luminescent signal will be reached in 10–30 minutes and will

have a half-life of several hours (Figure 3).

not use SDSas an activating agent for the gh a low

percentage of SDS is frequently used to monitor the chymotrypsin-like

activity of the proteasome, it is not necessary for activation in this

luminescent assay and is detrimental to the luciferase.

rd Assay (96-well, 100µl Final Reaction Volume)

50µl of Proteasome-Glo™ Reagent to each well of a white 96-well

plate containing 50µl of blank, control or test sample. If reusing tips, be

careful not to touch pipette tips to the wells containing samples to avoid

cross-contamination.

mix contents of wells using a plate shaker at 300–500rpm for

30 seconds. Incubate at room temperature for 10 minutes to 3 hours

depending upon convenience of reading time (Figure 3, Panel A). Maximal

signal is reached typically within 10–30 minutes using purified 20S

proteasome (Figure 3, Panel B). At this time, sensitivity is optimal.

Temperature fluctuations will affect the luminescent readings; if the room

temperature fluctuates too much, a constant-temperature incubator may

be desired.

luminescence with a plate-reading luminometer.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Part# TB349

Page 12

Printed in USA.

Revised 5/09

l Considerations

Sensitivity

The bioluminescent Proteasome-Glo™ Assays are more sensitive than

comparable fluorescent assays for several reasons. Biological samples can

contain naturally fluorescent compounds that contribute to background; the

luminescent assay eliminates background contributions from such compounds.

The luminescent substrates are not substrates for luciferase until they are

cleaved; hence, there is insignificant inherent background. Fluorescence

substrates generally depend on a shift in the excitation/emission wavelengths

after cleavage by the protease; consequently, there may be some overlap in the

emission spectra of the substrate before and after cleavage, creating substantial

inherent background. Pre-incubation of the luminescent proteasome substrates

with the Luciferin Detection Reagent insures that any contaminating free

aminoluciferin is consumed before beginning the assay (Figure 7). Any

contaminating free fluorophore remains in a fluorescent assay, contributing to

background. The low background also results in a broad linear range for the

luminescent assay (4 logs of proteasome concentration; Figures 2 and 4). The

assay sensitivity allows the researcher to use less proteasome if screening for

inhibitors. We recommend defining the linear range for the particular

proteasome preparation.

These coupled-enzyme assays are not dependent on accumulation of cleaved

product because the light output is a result of luciferase consuming the

aminoluciferin substrate as it is produced by the protease. Maximum sensitivity

is achieved as soon as the proteasome and luciferase activities reach a steady-

state. Typically this occurs in 10–30 minutes; therefore the assay is extremely

sensitive in a short time frame.

Note:Due to the sensitivity of the Proteasome-Glo™ Assays, contamination

with other luciferin-containing reagents can result in high background

luminescence. Be sure that shared luminometers are cleaned thoroughly before

performing this assay. Avoid workspaces and pipets that are used with

luciferin-containing solutions, including luminescence-based cell viability,

apoptosis or reporter gene assays.

Temperature and Signal Stability

Environmental factors that affect the rate of the luciferase reaction will also

affect the intensity of the light output and the stability of the luminescent

signal. Temperature can affect the rate of this enzymatic assay and thus the

light output. For consistent results, equilibrate assay plates to a constant

temperature before performing the assay. For batch-mode processing of

multiple plates, positive and negative controls should be included for each

plate. Additionally, precautions should be taken to ensure complete

temperature equilibration.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

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After rapidly reaching peak luminescence, the signal is relatively stable for

several hours (Figure 3). Ultimate signal stability may vary depending on the

proteasome preparation used.

Chemicals

The chemical environment of the luciferase reaction will affect the enzymatic

rate and thus luminescence intensity. Solvents used for various chemical

compounds may interfere with the luciferase reaction and thus the light output

from the assay. Dimethylsulfoxide (DMSO), commonly used as a vehicle to

solubilize organic chemicals, has been tested at final concentrations up to 1% in

the assay and found to have a minimal effect on light output.

Detergents

The Ultra-Glo™ Recombinant Luciferase is generally tolerant of a wide variety

of detergents. An exception is SDS, which will destroy the activity. Although a

low percentage of SDS is frequently used to assay the chymotrypsin-like

activity of the proteasome, it is not necessary for activation in this luminescent

assay and is detrimental to the luciferase.

nces

einer, M. and Hill, C.P. (2005) Mobilizing the proteolytic machine: Cell

biological roles of proteasome activators and inhibitors. Trends Cell Biol.15, 27–33.

ev, A.F., Kaganovich, D. and Goldberg, A.L. (2002) Binding of hydrophobic

peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites

of 20S proteasomes. J. Biol Chem.277, 22260–70.

ev, al.(2003) The caspase-like sites of proteasomes, their substrate

specificity, new inhibitors and substrates, and allosteric interactions with the trypsin-

like sites.J. Biol. Chem.278, 35869–77.

nover, A. (2005) Intracellular protein degradation: From a vague idea thru the

lysosome and the ubiquitin-proteasome system and onto human diseases and drug

targeting. Cell Death Diff.12, 1178–90.

es, P. M. et al.(2003) The proteasome as a target for cancer therapy. Clin.

Cancer Res.9, 6316–25.

n, D., Hideshima, T. and Anderson, K.C. (2005) Proteasome inhibition in

multiple myeloma: Therapeutic implication. Ann. Rev. Pharmacol. Toxicol.45, 465–73.

n, D. et al.(2005) A novel orally active proteasome inhibitor induces

apopotisis in multiple myeloma cells with mechanisms distinct from Bortezomib.

Cancer Cell8, 407–19.

, L.R. et al. (1996) Mechanistic studies on the inactivation of the proteasome by

lactacystin: A central role for clasto-lactacystin-β-lactone.J. Biol. Chem. 271, 7273–6.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Part# TB349

Page 14

Printed in USA.

Revised 5/09

d Products

Size

10ml

1

10ml

1

10ml

1

Cat.#

G8660

G8501

G8350

Protease Assays

Product

Proteasome-Glo™ Cell-Based Assay

Calpain-Glo™ Protease Assay

DPPIV-Glo™ Protease Assay

For Laboratory Use.

1

Additional Sizes Available.

Apoptosis Assays

Product

Caspase-Glo

®

8 Assay*

Caspase-Glo

®

9 Assay*

Caspase-Glo

®

3/7 Assay*

Apo-ONE

®

Homogeneous Caspase-3/7 Assay

DeadEnd™ Colorimetric TUNEL System

DeadEnd™ Fluorometric TUNEL System

*For Laboratory Use.

1

Additional Sizes Available.

Size

100ml

1

100ml

1

100ml

1

100ml

1

40 reactions

1

60 reactions

Cat.#

G8202

G8212

G8092

G7791

G7130

G3250

Cell Viability and Cytotoxicity Assays

ProductSize

CellTiter-Glo

®

Luminescent Cell Viability Assay (ATP)10ml

1

CellTiter-Blue

®

Cell Viability Assay (Resazurin)20ml

1

CellTiter 96

®

AQ

ueous

One Solution

Cell Proliferation Assay (MTS)*200 assays

1

CellTiter 96

®

AQ

ueous

Non-Radioactive

Cell Proliferation Assay (MTS)*1,000 assays

1

CellTiter 96

®

Non-Radioactive Cell

Proliferation Assay (MTT)*1,000 assays

1

MultiTox-Fluor Multiplex Cytotoxicity Assay*10ml

1

CytoTox-Fluor™ Cytotoxicity Assay*10ml

1

CytoTox 96

®

Non-Radioactive Cytotoxicity Assay (LDH)*1,000 assays

CytoTox-ONE™ Homogeneous Membrane

Integrity Assay (LDH)1,000–4,000 assays

1

*For Laboratory Use.

1

Additional Sizes Available.

Cat.#

G7570

G8080

G3582

G5421

G4000

G9200

G9260

G1780

G7891

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Printed in USA.

Revised 5/09

Part# TB349

Page 15

ADME Assays

Product

MAO-Glo™ Assay

P450-Glo™ CYP1A1 Assay

P450-Glo™ CYP1B1 Assay

P450-Glo™ CYP1A2 Screening System

P450-Glo™ CYP2C9 Screening System

P450-Glo™ CYP3A4 Screening System

P450-Glo™ CYP2D6 Screening System

P450-Glo™ CYP2C19 Screening System

Pgp-Glo™ Assay System

Pgp-Glo™ Assay System with P-glycoprotein

1

Additional Sizes Available.

Size

200 assays

1,000 assays

50ml

1

50ml

1

1,000 assays

1,000 assays

1,000 assays

1,000 assays

1,000 assays

10ml

10ml

Cat.#

V1401

V1402

V8752

V8762

V9770

V9790

V9800

V9890

V9880

V3591

V3601

Luminometers

Product

GloMax

®

96 Microplate Luminometer

GloMax

®

96 Microplate Luminometer

with Single Reagent Injector

GloMax

®

96 Microplate Luminometer

with Dual Reagent Injectors

Size

1 each

1 each

1 each

Cat.#

E6501

E6511

E6521

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Part# TB349

Page 16

Printed in USA.

Revised 5/09

(a)

Patent Pending.

(b)

U.S. Pat. Nos. 6,602,677 and 7,241,584, Australian Pat. Nos. 754312 and 785294, European Pat. No. 1131441 and other patents

pending.

(c)

The method of recombinant expression of Coleopteraluciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673

© 2006, 2009 Promega Corporation. All Rights Reserved.

Apo-ONE, Caspase-Glo, CellTiter 96, CellTiter-Blue, CellTiter-Glo, CytoTox 96 and GloMax are registered trademarks of

Promega Corporation. Calpain-Glo, CytoTox-Fluor, CytoTox-ONE, DeadEnd, DPPIV-Glo, MAO-Glo, P450-Glo, Pgp-Glo,

Proteasome-Glo, Suc-LLVY-Glo, Ultra-Glo, Z-LRR-Glo and Z-nLPnLD-Glo are trademarks of Promega Corporation.

GraphPad Prism is a registered trademark of GraphPad Software, Inc.

Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more

information.

All prices and specifications are subject to change without prior notice.

Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the

most up-to-date information on Promega products.

Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA

Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·

Printed in USA.

Revised 5/09

Part# TB349

Page 17