Thermo Fisher Scientific LanthaScreen Ubiquitin and SUMO Assay Reagents User guide

Brand: Thermo Fisher Scientific

Model: LanthaScreen Ubiquitin and SUMO Assay Reagents

Type: User guide

LanthaScreen™ Ubiquitin and SUMO Assay Reagents User Guide

Shipping Condition: Dry Ice Initial Storage: –20°C

Protocol part no. PV437X.pps Rev. date: 12 February 2008

For Technical Support for this or other Drug Discovery Products, dial 760-603-7200, option 3, extension 40266

Invitrogen Corporation • 5791 Van Allen Way • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

Table of Contents

1. LanthaScreen™ Ubiquitin and SUMO Products .........................................................................................................1

1.1 Ubiquitination Products.........................................................................................................................................................1

1.2 SUMOylation Products..........................................................................................................................................................1

2. Materials.........................................................................................................................................................................2

2.1 Ubiquitin and SUMO Reagents.............................................................................................................................................2

2.2 Additional Materials Needed.................................................................................................................................................2

2.3 Related Products ..................................................................................................................................................................2

3. Overview of TR-FRET ...................................................................................................................................................3

4. Instrument Settings ......................................................................................................................................................3

5. LanthaScreen™ Ubiquitination Assays .......................................................................................................................4

5.1 Example Assay Conditions ...................................................................................................................................................5

5.2 Example Assay Scheme.......................................................................................................................................................6

6. LanthaScreen™ SUMOylation Assays.........................................................................................................................6

7. Alternative Uses of LanthaScreen™ Ubiquitin and SUMO Reagents.......................................................................7

8. Assay Stability, Read Window, and Plate Selection..................................................................................................8

9. References.....................................................................................................................................................................9

10. Notice to Purchaser ......................................................................................................................................................9

1. LanthaScreen™ Ubiquitin and SUMO Products

Ubiquitin and ubiquitin-like proteins (Ubls) are broadly involved in key regulatory processes, including the proliferation of

cancer cells, and thus are an important set of potential drug targets. Traditional methods for analyzing ubiquitination—

including autoradiography, ELISA or immunoblotting—lack the necessary throughput to effectively identify modulators of

these events in a high-throughput screening (HTS) environment. LanthaScreen™ Ubiquitin and SUMO assay reagents were

specifically designed for use in HTS applications.

This user guide is intended as a reference when designing TR-FRET assays using LanthaScreen™ ubiquitin and SUMO

products. The guide provides typical assay conditions, formats, and example results for different types of assays.

1.1 Ubiquitination Products

LanthaScreen™ ubiquitination products provide HTS reagents to monitor changes in the rate of formation or the amount of

mono- and polyubiquitination of proteins. By incorporating the TR-FRET donor (terbium) and acceptor (fluorescein) onto the

ubiquitin itself, these universal assay reagents can be used to rapidly develop screening assays for ubiquitin-conjugating

enzymes. Due to the chemoselective labeling process, all of the lysines within ubiquitin are unmodified, and the labeled

reagents are readily incorporated onto ubiquitin-protein conjugates and into polyubiquitin chains.

1.2 SUMOylation Products

Ubiquitin-like proteins are small polypeptides (approx. 8−11 kDa) that covalently modify numerous intracellular proteins (via

a mechanism similar to ubiquitination) in order to mediate physiological and pathological responses in humans. Of these

Ubls, the best characterized are the SUMO family, including SUMO-1, SUMO-2, and SUMO-3. Modification by SUMO is

known to affect protein function by altering activity or cellular localization, modulating protein-protein interactions, or

competing for sites of ubiquitination (Johnson, 2004).

As with the ubiquitin reagents, LanthaScreen™ SUMO reagents have been chemoselectively labeled to ensure all of the lysines

within the ubiquitin-like proteins are unmodified. This allows the labeled reagents to be readily incorporated onto target

proteins or into poly-SUMO chains.

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2. Materials

2.1 Ubiquitin and SUMO Reagents

Reagents Size Cat. No.

LanthaScreen™ Tb-Ubiquitin 5 µg

25 µg

PV4375

PV4376

Fluorescein-Ubiquitin 50 µg

500 µg

PV4377

PV4378

Biotin-Ubiquitin 10 µg

100 µg

PV4379

PV4380

LanthaScreen™ Tb-SUMO-1 5 µg PV4717

Fluorescein-SUMO-1 50 µg PV4725

LanthaScreen™ Tb-SUMO-2 5 µg PV4719

Fluorescein-SUMO-2 50 µg PV4727

LanthaScreen™ Tb-SUMO-3 5 µg PV4721

Fluorescein-SUMO-3 50 µg PV4729

2.2 Additional Materials Needed

The following items are available from a variety of vendors:

• Conjugating enzymes E1, E2, and E3 (supplied as recombinant proteins or cell lysates)

• ATP

• EDTA

• A fluorescence plate reader with excitation capability at 340 nm and with the appropriate filters sets installed for

detecting the fluorescent emission signals of terbium at 495 nm and the acceptor fluor at 520 nm (see Section 4).

• Pipetting devices for 1–1000 µl volumes, suitable repeater pipettors, or multi-channel pipettors.

• Black 384-well assay plates. We recommend untreated polypropylene plates (e.g., MatriCal #MP101-1-PP with

total assay volume of 20-40 µl) or untreated polystyrene plates (e.g., Corning #3677 with total assay volume of

20 µl).

2.3 Related Products

Reagents Volume Cat. No.

LanthaScreen™ DUB Substrate 10 µg

100 µg

PV4443

PV4444

LanthaScreen™ De-SUMO-1 Substrate 25 µg PV4736

LanthaScreen™ De-SUMO-2 Substrate 25 µg PV4738

LanthaScreen™ De-SUMO-3 Substrate 25 µg PV4740

LanthaScreen™ De-NEDD8 Substrate 25 µg PV4742

LanthaScreen™ Tb-anti-ubiquitin-FK2 25 µg

1 mg

PV4752

PV4751

LanthaScreen™ TR-FRET Dilution Buffer 100 mL PV3574

LanthaScreen™ Tb-Streptavidin, 1 mg/mL 50 µg

1 mg

PV3576

PV3577

LanthaScreen™ Tb-anti-GST Antibody 25 µg

1 mg

PV4216

PV4217

For a complete, up-to-date listing of products, visit www.invitrogen.com/lanthascreen.

Invitrogen • LanthaScreen™ Ubiquitin and SUMO Assay Reagents User Guide Page 3 of 9

For Technical Support for this or other Drug Discovery Products, dial 760-603-7200, option 3, extension 40266

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Spectra of Terbium and Fluorescein

300 350 400 450 500 550 600 650

100

110

Fluorescein

Terbium

Wavelength (nm)

Excitation/Emission

3. Overview of TR-FRET

For screening libraries of compounds, time-resolved fluorescence resonance energy transfer (TR-FRET) is a recognized method

for overcoming interference from compound autofluorescence or light scatter from precipitated compounds. The premise of a

TR-FRET assay is the same as that of a standard FRET assay: when a suitable pair of fluorophores are brought within close

proximity of one another, excitation of the first fluorophore (the donor) can result in energy transfer to the second fluorophore

(the acceptor). This energy transfer is detected by an increase in the fluorescence emission of the acceptor, and a decrease in

the fluorescence emission of the donor. In HTS assays, FRET is often expressed as a ratio of the intensities of the acceptor and

donor fluorophores. The ratiometric nature of such a value corrects for differences in assay volumes between wells, and

corrects for quenching effects due to colored compounds.

In contrast to standard FRET assays, TR-FRET assays use a long-lifetime lanthanide chelate as the donor species. Lanthanide

chelates are unique in that their excited state lifetime (the average time that the molecule spends in the excited state after

accepting a photon) can be on the order of a millisecond or longer. This is in sharp contrast to the lifetime of common

fluorophores used in standard FRET assays, which are typically in the nanosecond range. Because interference from

autofluorescent compounds or scattered light is also on the nanosecond timescale, these factors can negatively impact

standard FRET assays. To overcome these interferences, TR-FRET assays are performed by measuring FRET after a suitable

delay, typically 50 to 100 microseconds after excitation, by a flashlamp excitation source in a microtiter plate reader. This

delay not only overcomes interference from background fluorescence or light scatter, but also avoids interference from direct

excitation due to the non-instantaneous nature of the flashlamp excitation source.

The most common lanthanides used in TR-FRET assays for HTS are terbium and europium. Terbium offers unique

advantages over europium when used as the donor species in a TR-FRET assay. In contrast to europium-based systems that

employ APC as the acceptor, terbium-based TR-FRET assays can use common fluorophores such as fluorescein as the

acceptor. In terbium-based TR-FRET assays, fluorescein-labeled reagents may be used rather than biotinylated molecules that

must then be indirectly labeled via streptavidin-mediated recruitment of APC as is commonly performed in europium-based

assays. The use of directly labeled molecules in a terbium-based TR-FRET assay reduces costs, improves kinetics, avoids

problems due to steric interactions involving large APC conjugates, and simplifies assay development, since there are fewer

independent variables requiring optimization in a directly labeled system.

4. Instrument Settings

The excitation and emission spectra of terbium and fluorescein are shown in Figure 1.

Figure 1: Excitation and Emission spectra of fluorescein and terbium.

As with other TR-FRET systems, the terbium donor is excited using a 340-nm excitation filter with a 30-nm bandwidth.

However, the exact specifications of the excitation filter are not critical, and filters with similar specifications will work well.

In general, excitation filters that work with europium-based TR-FRET systems will perform well with the LanthaScreen™

terbium chelates.

As shown in the figure, the terbium emission spectrum is characterized by four sharp emission peaks, with silent regions

between each peak. The first terbium emission peak (centered between approximately 485 and 505 nm) overlaps with the

maximum excitation peak of fluorescein. Energy transfer to fluorescein is then measured in the silent region between the first

two terbium emission peaks. Because it is important to measure energy transfer to fluorescein without interference from

terbium, a filter centered at 520 nm with a 25-nm bandwidth is used for this purpose. The specifications of this filter are more

critical than those of the excitation filter. In general, standard “fluorescein” filters may not be used, because such filters also

Invitrogen • LanthaScreen™ Ubiquitin and SUMO Assay Reagents User Guide Page 4 of 9

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pass light associated with the terbium spectra as well. The emission of fluorescein due to FRET is referenced (or “ratioed”) to

the emission of the first terbium peak, using a filter that isolates this peak. This is typically accomplished with a filter centered

at 490 or 495 nm, with a 10-nm bandwidth. In general, a 490-nm filter will reduce the amount of fluorescein emission that

“bleeds through” into this measurement, although instrument dichroic mirror choices (such as those on the Tecan Ultra

instrument) may necessitate the use of a 495-nm filter. The effect on the quality of the resulting measurements is minimal in

either case. Filters suitable for LanthaScreen™ assays are available from Chroma (www.chroma.com) as filter set PV001, or

from other vendors. A LanthaScreen™ filter module for the BMG LABTECH PHERAstar is available direct from BMG

LABTECH.

Aside from filter choices, instrument settings are typical to the settings used with europium-based technologies. In general,

guidelines provided by the instrument manufacturer can be used as a starting point for optimization. A delay time of 100 µs,

followed by a 200 µs integration time, would be typical for a LanthaScreen™ assay. The number of flashes or measurements

per well is highly instrument dependant and should be set as advised by your instrument manufacturer. In general,

LanthaScreen™ assays can be run on any filter-based instrument capable of time-resolved FRET, such as the Tecan Ultra, BMG

LABTECH PHERAstar, Molecular Devices Analyst, or PerkinElmer Envision. LanthaScreen™ assays have also been

performed successfully on the Tecan Safire2 monochromator-based instrument and the Molecular Devices M5 instrument.

Visit www.invitrogen.com/Lanthascreen or contact Invitrogen Discovery Sciences technical support at 800-955-6288 (select

option 3 and enter 40266), or email tech_support@invitrogen.com for more information on performing LanthaScreen™ assays

on your particular instrument.

5. LanthaScreen™ Ubiquitination Assays

Note: The experimental assay conditions for LanthaScreen™ SUMOylation assays are identical to those for LanthaScreen™

ubiquitination assays, with only a few modifications. See Section 6.

For a typical HTS TR-FRET ubiquitination assay, the target protein to be ubiquitinated is combined with ubiquitin-conjugating

enzymes (E1, E2, and E3), ATP, and the appropriate LanthaScreen™ ubiquitin analog(s). The ubiquitin-conjugating enzymes

can be supplied as purified reagents or as crude cellular lysate that overexpresses or contains sufficient quantities of your

conjugation enzymes (i.e., HeLa Fraction I or Fraction II). During the ubiquitination reaction, the conjugation enzymes attach

the labeled ubiquitin(s) onto the target protein, resulting in mono- or polyubiquitination. Depending upon the specific assay

format, a detection reagent (i.e., LanthaScreen™ Tb-anti-epitope tag antibody or LanthaScreen™ Tb-Streptavidin) is added to

the reaction to complete the homogenous TR-FRET assay. See Figure 2.

Figure 2. Graphical representation of the LanthaScreen™ HTS ubiquitination assay

formats. For the ubiquitination of an epitope-tagged target protein (A), a LanthaScreen™

Tb-labeled anti-epitope antibody is required to complete the TR-FRET pairing. Since the

terbium is supplied by a labeled antibody, this assay format can detect both mono-

ubiquitination (a single site of modification) and polyubiquitination (a polyubiquitin

chain) of the target protein. When a primary antibody towards your target protein (or an

epitope tag) is not available, the Intrachain (B) (TR-FRET donor: LanthaScreen™ Tb-

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ubiquitin) or Biotin/Streptavidin (C) (TR-FRET donor: LanthaScreen™ Tb-Streptavidin

(SAv)) ubiquitination assay formats can be used. Since both the TR-FRET donor and

acceptor (fluorescein-ubiquitin) are present in the polyubiquitin chain in the Intrachain

assay, no development step is required. This makes the Intrachain assay especially useful

when real-time kinetics information on ubiquitination is desired.

The extent of target-protein ubiquitination is directly related to the TR-FRET signal. In general, an increase in the TR-

FRET signal signifies the ubiquitination of the target protein, whereas no increase in the TR-FRET signal would

suggest that the target protein is not ubiquitinated. In HTS applications, a compound is introduced to measure the

effectiveness of the compound to inhibit or promote ubiquitination of the target protein. If the compound inhibits the

ubiquitination reaction, a decrease in the TR-FRET signal (compared to control wells) would be observed due to a

decrease in the ubiquitination of the target protein. Conversely, an increase in the TR-FRET signal would be

observed if the compound promotes the ubiquitination of the target protein.

LanthaScreen™ Tb-labeled anti-species antibodies provide additional assay options for detecting ubiquitination when

a primary antibody to the target protein is available. The versatility of the LanthaScreen™ ubiquitination reagents

allows the user to easily construct a custom assay solution that will integrate the advantages of TR-FRET HTS into

your current biochemical system with minimal development time.

5.1 Example Assay Conditions

All LanthaScreen™ ubiquitin assays use similar assay conditions and only vary in which LanthaScreen™ assay

reagent(s) or detection reagents that are required. Table 1 outlines example assay conditions for the LanthaScreen™

TR-FRET ubiquitination assays.

Note: These assay parameters are supplied as a starting point for further assay optimization to ultimately achieve

the best possible assay signal for your specific biochemical system.

For endpoint analysis, ATP hydrolysis can be halted with the addition of EDTA at a concentration equal to (or

slightly above) the Mg2+ concentration within the reaction.

Table 1. Example concentrations for LanthaScreen™ ubiquitination assay components

Final Concentration Range in Reaction

Reagents Anti-Epitope Assay Intrachain Assay Biotin/Streptavidin Assay

Assay Buffer (50 mM Tris-HCl pH 8,

1 mM DTT†, and 4 mM MgCl2) — — —

ATP‡ 1 mM 1 mM 1 mM

Proteins/Conjugating Enzymes††

Target Protein 10–50 nM 10–50 nM 10–50 nM

E1 10–50 nM 10–50 nM 10–50 nM

E2 50–200 nM 50–200 nM 50–200 nM

E3 50–200 nM 50–200 nM 50–200 nM

LanthaScreen™ Ubiquitin Reagent

Fluorescein-ubiquitin 350 nM 300 nM 300 nM

Tb-ubiquitin — 25 nM —

Biotin-ubiquitin — — 100 nM

Detection/Stop Reagents

EDTA‡‡ 10 mM 10 mM 10 mM

LanthaScreen™ Tb-labeled Antibody 2–10 nM — —

LanthaScreen™ Tb-Streptavidin — — 10 nM

† The addition of DTT is optional

‡ An "ATP Regeneration System" outlined by Yao et al. (Yao & Cohen, 2000) can also be used

†† Conjugating enzymes can be supplied as purified reagents or as crude cellular lysate preparations.

‡‡ The addition of EDTA is only required if end point analysis is desired

For detailed step-by-step instructions and example data for all of the LanthaScreen™ ubiquitination assays, please

visit www.invitrogen.com/Lanthascreen or contact technical support at 1-800-955-6288, option 3, extension 40266, or

email [email protected].

Invitrogen • LanthaScreen™ Ubiquitin and SUMO Assay Reagents User Guide Page 6 of 9

For Technical Support for this or other Drug Discovery Products, dial 760-603-7200, option 3, extension 40266

Invitrogen Corporation • 5791 Van Allen Way • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

5.2 Example Assay Scheme

A general assay scheme for the LanthaScreen™ ubiquitination assays is outlined below and in Figure 3. Exact assay

volumes and incubation times can be modified and should be optimized to identify the best possible assay window

for your biochemical system.

1. Prepare a 2X Ubiquitination Master Mix, containing E1, E2, and E3 enzymes, target protein, and

LanthaScreen™ Ubiquitination reagent(s).

2. Add 2.5 µL of a 4X inhibitor, test compound, or assay buffer to the well.

3. Add 5 µL of the 2X Ubiquitination Master Mix to the well.

4. Add 2.5 µL of 4X ATP solution to start the reaction.

5. Incubate for 1–6 hours.

6. Add 5 µL of 3X Detection/Stop Reagent (containing EDTA and LanthaScreen™ Tb-labeled antibody or

LanthaScreen™ Tb-Streptavidin) to the well.

7. Equilibrate for 30–60 minutes then read on an appropriate plate reader.

Figure 3. General assay scheme for the LanthaScreen™ ubiquitination assays.

6. LanthaScreen™ SUMOylation Assays

As with the ubiquitin reagents, the LanthaScreen™ SUMO reagents have been chemoselectively labeled to ensure all of the

lysines within the ubiquitin-like proteins are unmodified. This allows the labeled reagents to be readily incorporated onto

target proteins or into poly-SUMO chains. The experimental assay conditions for LanthaScreen™ SUMOylation assays are

identical to those for the ubiquitination assay reagents (see Section 5), with only a few modifications as shown in Table 2.

Invitrogen • LanthaScreen™ Ubiquitin and SUMO Assay Reagents User Guide Page 7 of 9

For Technical Support for this or other Drug Discovery Products, dial 760-603-7200, option 3, extension 40266

Invitrogen Corporation • 5791 Van Allen Way • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

Table 2. Example concentrations for LanthaScreen™ SUMOylation assay components

Final Concentration Range in Reaction

Reagents Anti-Epitope Assay Intrachain Assay

Assay Buffer (50 mM Tris-HCl pH 7.5, 1

mM DTT†, and 4 mM MgCl2) — —

ATP‡ 1 mM 1 mM

Proteins/Conjugating Enzymes††

Target Protein 10–50 nM 10–50 nM

E1 5–10 nM 5–10 nM

E2 50–200 nM 50–200 nM

E3 50–200 nM 50–200 nM

LanthaScreen Ubiquitin Reagent

Fluorescein-SUMO-1/2/3 200 nM 50 nM

Tb-SUMO-1/2/3 — 5 nM

Detection/Stop Reagents

EDTA‡‡ 20 mM 20 mM

LanthaScreen™ Tb-labeled Antibody 2–10 nM —

† The addition of DTT is optional

‡ An "ATP Regeneration System" outlined by Yao et al. (Yao & Cohen 2000) can also be used

†† Conjugating enzymes can be supplied as purified reagents or as crude cellular lysate

preparations.

‡‡ The addition of EDTA is only required if end point analysis is desired

For detailed step-by-step instructions on how to perform the LanthaScreen™ SUMOylation assays, as well as example

experimental data on the SUMOylation of RanGAP, please visit www.invitrogen.com/Lanthascreen or contact Invitrogen

Discovery Sciences technical support at 800-955-6288 (select option 3 and enter 40266), or email [email protected].

7. Alternative Uses of LanthaScreen™ Ubiquitin and SUMO Reagents

The versatility of the LanthaScreen™ ubiquitin and SUMO reagents allows one to mix and match reagents to develop

customizable assay solutions. For example, to develop a LanthaScreen™ substrate for a de-SUMOylation enzyme (or

deubiquitinating enzyme) of interest, Fluorescein-SUMO-1/2/3 (or fluorescein-ubiquitin) can be enzymatically attached to a

target protein containing an epitope tag (i.e., GST) using the assay conditions outlined above. Following a quick purification

step (i.e., GSH agarose or other similar chromatography step), you can obtain a specific SUMOylated substrate to assay de-

SUMOylation activity. See Figure 3.

Invitrogen • LanthaScreen™ Ubiquitin and SUMO Assay Reagents User Guide Page 8 of 9

For Technical Support for this or other Drug Discovery Products, dial 760-603-7200, option 3, extension 40266

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Figure 3. Customized deconjugation substrates can be developed using the

LanthaScreen™ ubiquitin and ubiquitin-like reagents. For example, the SUMOylation of

RanGAP1 with Fluorescein-SUMO-1 results in a customized deconjugation substrate that

can be cleaved by SENP1. Substrate cleavage is detected with the addition of a

LanthaScreen™ Tb-anti-GST antibody and is directly related to a decrease in the TR-FRET

signal.

*Time saving tip* To speed up your immunoblotting experiments, both the fluorescein-ubiquitin and SUMO reagents can be

used to visualize ubiquitinated (or SUMOylated) products in the protein gel without the need to perform time-consuming

immunoblotting transfer steps. Following electrophoresis, the protein gel can be immediately visualized on a fluorescence gel

scanner using the appropriate instrument settings for fluorescein.

8. Assay Stability, Read Window, and Plate Selection

For a given assay system, signal stability and the read window should be assessed. In general, assays showed a stable signal

for 12 hours following the development step. While some experiments have shown the development of a TR-FRET signal

with incubation times as little as 30 minutes, other systems may require 3-6 hours to develop a signal. Depending on the

specific assay configuration and the demands of the assay, incubation times may vary and should be determined

experimentally for the given assay system. While we recommend black Corning® 384-well, low-volume, round-bottom (non-

binding surface) assay plates (#3676). Other low volume assay plates, while not tested, may be suitable.

Invitrogen • LanthaScreen™ Ubiquitin and SUMO Assay Reagents User Guide Page 9 of 9

For Technical Support for this or other Drug Discovery Products, dial 760-603-7200, option 3, extension 40266

Invitrogen Corporation • 5791 Van Allen Way • Carlsbad, CA 92008 • Phone: 760 603 7200 • FAX: 760 602 6500 • www.invitrogen.com

9. References

1. Johnson, E. S. 2004. Protein modification by SUMO, Annu. Rev. Biochem., 73, 355−382

2. Yao, T., and Cohen, R. E. 2000. Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme. J. Biol. Chem., 275, 36862-

36868.

10. Notice to Purchaser

Limited Use Label License No. 176: Lanthanide Chelates

This product is the subject of one or more of US Patents 5,622,821 5,639,615, and 5,656,433 and foreign equivalents. The

purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and

components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The

buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its

components to a third party or otherwise use this product or its components or materials made using this product or its

components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product

to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in

writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information

solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration

and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its

components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or

prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are

resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above

patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product

developed in research by the buyer in which this product or its components was employed, provided that neither this product

nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the

limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For

information on purchasing a license to this product for purposes other than research, contact Licensing Department,

Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.

Limited Use Label License No. 178: Lifetime-resolved assay procedures

This product is sold under license to U.S. Patent 4,822,733 from Vysis, an Abbott Laboratories company. This product is

licensed for research use only and may not be used for in vitro diagnostics.

The performance of this product is guaranteed for six months from the date of purchase if stored and handled properly.

This product and/or the use of this product is covered under one or more of the following US patents:

5,622,821, 5,639,615, 5,656,433, and 4,822,733, as well as pending patent applications owned by or licensed by Invitrogen Corporation.

Thermo Fisher Scientific LanthaScreen Ubiquitin and SUMO Assay Reagents User guide

Thermo Fisher Scientific LanthaScreen Ubiquitin and SUMO Assay Reagents User guide

Thermo Fisher Scientific LanthaScreen Ubiquitin and SUMO Assay Reagents User guide

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