Read Cellular Protein Fractionation Kit text version

PerkinElmer Life and Analytical Sciences, Inc.

CELLULAR PROTEIN FRACTIONATION KIT CATALOG NUMBER PRD101A001KT

For Laboratory Use CAUTION: Research Chemicals for Research Purposes

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Table of contents

I. II. III. IV. V. VI. VII. A. B. C. VIII. IX. X. XII. XII. Product name Intended use Introduction Principles of the procedure Kit components, storage and stability Additional materials and equipment required but not supplied Assay protocol Subcellular extraction of proteins from adherent tissue culture cells Subcellular extraction of proteins from suspension-grown tissue culture cells Subcellular extraction of proteins from fragmented tissue and frozen cell pellets Troubleshooting guide Safety considerations References Licensing Name and place of manufacture 4 4 4 6 9 10 10 11 14 17 19 20 21 21 21

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I.

PRODUCT NAME

CELLULAR PROTEIN FRACTIONATION KIT Cat. # PRD101A001KT

II.

INTENDED USE

The Cellular Protein Fractionation Kit is designed for the subcellular extraction of mammalian proteins from the cytosolic, membrane / organelle, nuclear, and cytoskeletal fractions of adherent tissue culture cells, suspension tissue culture cells, frozen cell pellets, and fragmented tissues.

For Laboratory Use Caution: Research Chemicals for research purposes only.

III. INTRODUCTION

Proteins redistribute in response to a variety of physiological stimuli. Activation of many cellular regulatory pathways is accompanied by the translocation of key proteins from one region of the cell to another. Additionally, clustering of plasma membrane receptors by an extracellular ligand often leads to the association of specific integral transmembrane proteins with the underlying cytoskeleton. Despite their obvious regulatory significance, dynamic changes in protein compartmentalization have not routinely been monitored quantitatively by combining selective plasma membrane labeling, subcellular fractionation and PAGE. One major challenge in functional proteomics is the separation of complex protein mixtures to allow detection of low abundance proteins and provide for quantitative and qualitative analysis of proteins impacted by environmental parameters. Prerequisites for the success of such analysis include standardized and reproducible procedures for sample preparation prior to 1-D or 2-D gel electrophoresis and/or Mass Spectrometry. Due to the complexity of total proteomes and the divergence of protein properties, it is necessary to prepare standardized partial proteomes of a given cell in order to maximize the coverage of the proteome and to increase the probability of visualizing low-abundance proteins.

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With the PerkinElmer Cellular Protein Fractionation Kit, eukaryotic cells are fractionated into four distinct protein extracts according to their subcellular localization: cytosolic, membrane/organelle, nuclear and cytoskeletal. The method employs detergents to sequentially extract proteins from small amounts of starting material. The kit takes advantage of the differential solubility of proteins in various subcellular compartments. It utilizes highly specialized extraction buffers to target specific subcellular compartments and simultaneously preserve the structural integrity of the proteins before and during each sequential extraction. A schematic overview of the extraction procedure applied to adherent tissue culture cells is shown in Figure 1, highlighting the morphological changes of the cells. For adherent cells, the sequential extraction is performed directly in the tissue culture dish without removing the cells. At each step of the extraction procedure the insoluble cellular fractions remain attached to the plate, until the appropriate extraction reagent is applied. For suspension-grown cells, extraction starts with gentle sedimentation and washing of the cells. The stepwise extraction delivers all four protein fractions from a single specimen. As a result, the early destruction of the cellular structure by enzymatic or mechanical detachment of cells from the tissue culture plate and any mixing of different subcellular compartments is prevented. The specialized, mild procedure yields the majority of proteins in their native state, making it highly suitable for demanding proteomics applications, including enzyme activity assays (e.g. reporter gene assays) and subcellular redistribution assays to monitor protein shuttling (e.g. signaling proteins).

Figure 1. Adherent SAOS cells were extracted according to the detailed protocol for Subcellular Extraction of Proteins from Adherent Cells as outlined above. Images i-iv shows the morphology of the cells before and after each extraction step (200-fold enlarged). The SAOS cells remained attached throughout the extraction procedure.

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Fractionation by sequential extraction of cells or tissues with detergentcontaining buffers allows partitioning of proteins into functionally distinct compartments readily evaluated by 1-D or 2-D gel electrophoresis. Unlike most other subcellular fractionation methods, detergent fractionation preserves the integrity of the cytoskeletal compartment. The regulatory significance of the cytoskeletal compartment is increasingly recognized in processes such as endocytosis, exocytosis, mitosis, cytokinesis, chemotaxis, signal transduction, and protein biosynthesis. Differential detergent extraction is simple, independent of timeconsuming ultracentrifugation or cumbersome washing steps, amenable to low quantities of cells and fully compatible with commonly employed electrophoretic procedures. PerkinElmer also offers an Iminobiotin Protein Labeling Kit (Cat. # PRD100A001KT) that can be used in combination with the Cellular Protein Fractionation Kit in order to monitor protein compartmentalization. Labeling plasma membrane proteins with iminobiotin selectively highlights plasma membrane proteins in the various detergent fractions, and provides the opportunity to enrich these proteins further using subsequent affinity isolation procedures employing immobilized streptavidin or avidin.

IV. PRINCIPLES OF THE PROCEDURE

The Cellular Protein Fractionation procedure yields the total proteome fractionated into four sub-proteomes of decreasing complexity. Using Cytosol Buffer, the cytosolic proteins are released. In the second step the membrane and organelle proteins are solubilized using Membrane / Organelle Buffer. Nuclear Buffer yields the nuclear proteins. In the final step,using Cytoskeletal Buffer,the components of the cytoskeleton are solubilized. The efficiency of the subcellular fractionation by the kit has been shown by 1-D PAGE (Figure 2A) and immunoblotting of selected marker proteins (Figure 2B). More than 90% of subcellular marker proteins can be assigned to the expected subcellular fraction. Please note that HSP70 is present in both the cytoplasm and the mitochondria of cells and is thus detected in both the cytoplasmic and membrane/organelle fractions using the Cellular Protein Fractionation procedure. The Cellular Protein Fractionation Kit has been used successfully with a wide variety of human tissue culture cells. Table 1 below lists the quantity of protein obtained in each fraction following

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Cellular Protein Fractionation Kit extraction (The cells were grown in T25 tissue culture flasks with approximately 1 x 106 cells/flask at approximately 80% confluence. Please note that the actual number of cells at 80% confluency may differ considerably among different cell types).

Table 1. Protein Concentrations of Each Fraction Obtained From Various Cells

Cell type (human)

Fraction

[Protein] (mg/ml)

Protein amount (mg) 0.55

Proportion to total protein (mg) 33%

Deviation (%)

Cytosol Epithelial carcinoma A431 Membrane/ Organelle Nucleus Cytoskeleton Cytosol Mammalian carcinoma MCF7 Membrane/ Organelle Nucleus Cytoskeleton Cytosol Liver carcinoma HEPG2 Membrane/ Organelle Nucleus Cytoskeleton Cytosol Membrane/ Organelle Nucleus Cytoskeleton

0.55

<10

0.75 0.2 0.15 0.64

0.75 0.1 0.075 0.64

45% 13% 9% 44%

<10 <10 <5 <10

0.47 0.24 0.1 3.1

0.47 0.12 0.05 3.1

32% 17% 7% 53%

<10 <10 <5 <10

1.8 0.6 0.35 0.44

1.8 0.3 0.175 0.44

31% 10% 6% 36%

<10 <10 <5 <10

Osteosarcom SAOS2

0.5 0.2 0.1

0.5 0.1 0.05

41% 17% 6%

<10 <10 <5

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The Cellular Protein Fractionation Kit procedure is ideally suited for investigating changes in subcellular localization of regulatory proteins under certain experimental conditions or in certain disease states. To demonstrate this application, the translocation of NF-B from the cytosol to the nucleus upon stimulation of cells with tumor necrosis factor (TNF-) (Mejdoubi et al. 1999; Butcher et al. 2001) was chosen as a model system. NF-B translocation was studied in TNF--stimulated A431 cells that were subsequently extracted with the Cellular Protein Fractionation Kit. Nuclear translocation of NF-B is easily demonstrated by immunoblotting of the 4 fractions and by densitometric analysis of filters (Figure 3). The control protein, calpain, did not undergo any translocation among fractions following TNF--stimulation of cells (data not shown). Thus, using the Cellular Protein Fractionation Kit procedure, translocation of regulatory proteins can be investigated.

A

B

Figure 2. Adherent SAOS cells were fractionated according to the Detailed Protocol for Subcellular Extraction of Proteins into Cytosol (1); Membrane/Organelle (2); Nuclear (3); and Cytoskeleton (4) as outlined. A: An aliquot of each fraction from were subjected to SDS-PAGE analysis. The data demonstrate clear differences in the protein banding patterns among the 4 fractions. B: Aliquots of each fraction were separated by SDS-PAGE and transferred to PVD membrane for blotting with the indicated antibodies. For c-Jun, the fractions were subjected to immunoprecipitation, prior to Western blotting, to enrich for any c-Jun present in each fraction. The data demonstrate that each marker protein is specifically enriched within the appropriate fraction.

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cytosol 80 60 40 20 0

membrane/organelle

nucleus

cytoskeleton

0

5

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Figure 3. Analysis of Protein Distribution Profiles - Translocation of NF-B. A431 cells were stimulated with 0.2 µg/ml TNF- for the indicated times. At the end of each induction period the cells were extracted as outlined in the Detailed Protocol for Subcellular Extraction of Proteins from Adherent Cells. The proteins from an aliquot of each fraction were separated by SDS-PAGE and transferred to PVDF membrane for Western blot analysis using an antibody specific for NF-B. The data indicate that there is measurable translocation of NFB from the cytoplasm to the nucleus as early as 5 minutes after TNF- stimulation.

V. KIT COMPONENTS, STORAGE AND STABILITY

Note: Upon arrival store the components of the kit as specified below. Prior to performing the extraction protocol, all frozen buffers must be thawed at room temperature. A water bath set at 25 °C will aid in the thawing process. Mix the buffers well by gentle shaking or vortexing.

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The Cellular Protein Fractionation kit contains the following components: 200 mL 2 X 22 mL 44 mlL 22 mL 1 mL 20 mlL 90 µL Wash Buffer Cytosol Buffer Membrane/Organelle Buffer Nuclear Buffer Protease Inhibitor Cocktail Cytoskeletal Buffer * Nuclease **

Storage: Other than the two components specified below, store all other components at 2-8 °C. For long-term storage, aliquot and freeze at -20° C. Avoid freeze / thaw cycles. * Cytoskeletal Buffer can be stored at RT on bench top or shelf. ** Nuclease can be stored at 2-8 °C and must be kept on ice during use.

VI. ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED BUT NOT SUPPLIED

Platform mixer and rotary shaker Cooled centrifuge and rotor for 50 ml tube size Cooled micro centrifuge and rotor up to 10,000 x g for 2 ml tube size Thermo mixer or rolling facility

VII. ASSAY PROTOCOL

Appropriate assay samples types include: Adherent tissue culture cells Suspension-grown tissue culture cells Frozen cell pellets Fragmented tissue

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1. Before beginning the extraction, mix the Wash Buffer and all Extraction Buffers well by vortexing. During the extraction keep Cytosol Buffer, Membrane/Organelle Buffer, Nuclear Buffer, and Nuclease on ice and Cytoskeletal Buffer and Protease Inhibitor Cocktail at room temperature. The buffers must be completely thawed before starting the extraction. 2. Carefully remove the cell culture medium without disturbing the cell monolayer. 3. Wash the cell culture flasks containing the cell monolayers twice with 2 ml of the Wash Buffer for every 25 cm2 of cell surface area (12 ml for T-150, 6 ml for T-75, etc.). For optimal adherence of the cells during the extraction procedure, it is vital that the cells are in the logarithmic phase of growth at ~ 80% confluency. Wash the cells by gently mixing and removing the wash solution. 4. Add 1.0 ml of Cytosol Buffer and 3.0 µl Protease Inhibitor Cocktail for every 25 cm2 of cell surface area (6 ml for T-150, 3 ml for T-75, etc.) directly to each flask. Position flasks so that the reagents are in contact with the cells. 5. Incubate the flask at 4 °C for 10 minutes with constant rocking. 6. Remove Cytosol Buffer from the flask and set aside at 4 °C in an appropriate polypropylene tube. This solution is enriched in the cell's soluble cytosolic proteins. 7. Add 1 ml of ice-cold Membrane/Organelle Buffer and 3.0 µl Protease Inhibitor Cocktail for every 25 cm2 of cell surface area (6 ml for T-150, 3 ml for T-75, etc.) to the flask. Position flasks so that the reagents are in contact with the cells. Incubate the flask at 4 °C for 30 minutes with constant rocking. 8. Using a pipette, transfer the supernatant (membrane/organelle fraction) to a clean tube; take care not to disturb the remaining cell monolayer. Make sure that all liquid is removed. Store the membrane/organelle proteins on ice.

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9.

10. 11.

12.

13.

14.

Mix 500 µl ice-cold Nuclear Buffer with 5 µl Protease Inhibitor Cocktail and 1.5 µl Nuclease for every 25 cm2 of cell surface area (3 ml for T-150, 1.5 ml for T-75, etc.) to the flask and immediately add the mixture to the flask/dish without disturbing the cell monolayer. Carefully rotate the flask/dish until all cells are covered. Incubate for 10 minutes at 4 °C under gentle agitation (Note: if the cells detach, transfer the cells in the Wash Buffer to an appropriate centrifuge tube). Using a pipette, transfer the supernatant (Nuclear proteins) to a clean tube; take care not to disturb the remaining cell monolayer. Completely remove all the liquid. Store the nuclear proteins on ice. Mix 500 µl RT Cytoskeletal Buffer with 5 µl Protease Inhibitor Cocktail for every 25 cm2 of cell surface area (3 ml for T-150, 1.5 ml for T-75, etc.). Immediately add the mixture to the flask/dish. Carefully rotate the flask/dish until all remaining cell components are covered. The remaining cell structures will detach upon treatment with Cytoskeletal Buffer. After complete solubilization of the residual materials, transfer the extract (cytoskeletal proteins) to a clean tube. Store the cytoskeletal fraction on ice. (Please refer to the Technical Appendix). If the fractions are to be used immediately, continue to store on ice prior to performing any downstream applications and analyses. For long-term storage, dispense each fraction into aliquots (e.g., 100 µl) and freeze at -20 °C or -70 °C until use. For use of the fractions for 1- or 2-D gel electrophoresis, refer to the TROUBLESHOOTING GUIDE.

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1. Before beginning the extraction, mix the Wash Buffer and all Extraction

Buffers well by vortexing. During the extraction keep Cytosol Buffer, Membrane/Organelle Buffer, Nuclear Buffer, and Nuclease on ice and Cytoskeletal Buffer and Protease Inhibitor Cocktail at room temperature. The buffers must be completely thawed before starting the extraction. 2. Transfer the cells to an appropriate centrifuge tube and pellet by centrifugation at 100-300 x g, room temperature for 10 minutes. Aspirate and discard the supernatant without disturbing the cell pellet. 3. Add 4 ml Wash Buffer (room temperature) and suspend the cell pellet by gently pipetting up and down. 4. Pellet the cells by centrifugation at 100-300 x g, 4 °C for 10 minutes. Carefully remove and discard the supernatant without disturbing the cell pellet. 5. Repeat steps 3 and 4 to ensure complete removal of contaminating medium. 6. Mix 1 ml of ice cold Cytosol Buffer and 5 µl Protease Inhibitor Cocktail per 3x106 cells, then add the mixture directly to the cell pellets. 7. Suspended the cell pellet by gently pipetting up and down and incubate the tubes at 4 °C for 10 minutes on a rocking platform shaker. 8. Pellet the insoluble material by centrifugation at 500-1000 x g, 4 °C for 10 minutes. Using a pipette, transfer the supernatant (cytosol proteins) to a clean tube. Store the cytosol fraction on ice. This fraction is enriched in the cell's soluble cytosolic proteins. 9. Add Membrane/Organelle Buffer (1 ml of buffer and 5 µl Protease Inhibitor Cocktail per 3x106 cells; premix). Suspend the cell pellet by gently pipetting up and down. Incubate at 4 °C for 30 minutes on a rocking platform shaker to avoid formation of cell clumps.

10. Pellet the insoluble material by centrifugation at 5000-6000 x g, 4 °C for 10 minutes. 11. Using a pipette, transfer the supernatant (membrane/organelle proteins) to a clean tube. Store the membrane/organelle proteins on ice. 12. Mix 500 µl Nuclear Buffer with 5 µl Inhibitor Cocktail and 1.5 µl Nuclease per 3x106 cells and immediately add the mixture to the cell pellet. Resuspend the cell pellet by pipetting up and down. Incubate the tubes for 10 minutes at 4 °C under gentle agitation. Use of a rotary shaker is recommended to avoid formation of cell clumps

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13. Pellet the insoluble material by centrifugation at 6800 x g, 4 °C for 10 minutes. 14. Using a pipette, transfer the supernatant (Nuclear proteins) to a clean tube. Store the nuclear fraction on ice. 15. Mix 500 µl room temperature Cytoskeletal Buffer with 5 µl Protease Inhibitor Cocktail per 3x106 cells and immediately add the mixture to the pellet. Carefully suspend residual particles by pipetting up and down (cytoskeletal proteins). 16. If the fractions are to be used immediately, continue to store them on ice prior to performing any downstream applications and analyses. For longterm storage, dispense each fraction into aliquots (e.g., 100 µl) and freeze at -20 °C or -70 °C until use. 17. For use of the fractions for 1- or 2-D gel electrophoresis, refer to the TROUBLESHOOTING GUIDE.

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1. Mix 1 ml ice-cold Cytosol Buffer with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the fragmented tissue or frozen cell pellet. Gently resuspend the fragmented tissue or frozen cell pellet by gently pipetting up and down. Incubate at 4 °C for 10 minutes under gentle agitation. The use of a rotary shaker is recommended to avoid formation of cell clumps. 2. Pellet the insoluble material by centrifugation for 10 minutes at 500-1000 x g, 4 °C. 3. Carefully transfer the supernatant (Cytosol) to a clean tube. Store the cytosol proteins on ice. 4. Mix 1 ml ice-cold Membrane/Organelle Buffer with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the pellet. Resuspend the pellet by gently pipetting. Incubate the sample at 4 °C for 30 minutes under gentle agitation. Use of a rotary shaker is recommended to avoid formation of cell clumps. 5. Pellet the insoluble material by centrifugation at 5000-6000 x g, 4 °C for 10 minutes. 6. Carefully transfer the supernatant (Membrane / Organelle proteins) to a clean tube. Store membrane/organelle proteins on ice. 7. Mix 500 µl Nuclear Buffer with 5 µl Inhibitor Cocktail and 1.5 µl Nuclease and immediately add the mixture to the pellet. Suspend the pellet by gently pipetting up and down. Incubate the sample at 4 °C for 10 minutes under gentle agitation. Use of a rotary shaker is recommended to avoid formation of cell clumps. 8. Pellet the insoluble material by centrifugation at 7000 x g, 4 °C for 10 minutes. 9. Carefully transfer the supernatant (Nuclear) to a clean tube. Store the nuclear proteins on ice. 10. Mix 500 µl room temperature Cytoskeletal Buffer with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the pellet. Suspend the residual particles by pipetting them up and down (Cytoskeletal proteins). 11. If the fractions are to be used immediately, continue to store them on ice prior to performing any downstream applications and analyses. For longterm storage, dispense each fraction into aliquots (e.g., 100 µl) and freeze at -20 °C or -70 °C until use. 12. For use of the fractions for 1- or 2D gel electrophoresis, refer to the TROUBLESHOOTING GUIDE.

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VIII. TROUBLESHOOTING GUIDE

Upon centrifugation of suspension-grown cells, no compact pellet is formed. It cannot be ruled out that certain types of cells do not form compact pellets using an acceleration of 100 x g. In these cases increase the acceleration of the centrifuge to up to 300 x g.

Upon washing or extraction of adherent tissue culture cells, detachment of the cell monolayer occurs. When adherent cell detach during the extraction steps transfer the resulting cell suspension to a clean tube and continue with the extraction respective step using the Detailed Protocol for Subcellular Extraction of Proteins from Suspension-Grown Cells. This does normally not affect the quality of the results.

How do I determine the protein concentration of the subcellular protein extracts? As the extraction buffers contain components that might interfere with protein quantification assays specific protein assays such as the EZQTM Protein Quantitation Assay (Invitrogen Cat. No. R3320) are required to determine the protein concentration.

When storing Cytoskeleton Buffer or Cytoskeleton fractions on ice, a precipitate occurs. This does not normally affect the results of the extraction or downstream experiments. If a precipitate forms in Cytoskeleton Buffer or Cytoskeleton Buffer fractions, gently warm the sample to room temperature, mix well, and use immediately for extraction or analysis, respectively. How do I prepare subcellular fractions generated with the Cellular Protein Fractionation Kit for 1-D SDS-PAGE? The Cellular Protein Fractionation Kit fractions can be directly analyzed by one-dimensional SDS-PAGE: Dilute the sample with an equal volume of 2X SDS-PAGE sample buffer (e.g., 125 mM Tris-HCl, pH 6.8; 10% (w/v) SDS; 30% (v/v) glycerol; 100 mM DTT; 0.002% (w/v) bromophenol blue) and heat to 95 °C for 5 minutes prior to loading the gel.

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How can I concentrate the protein fractions following use of the Cellular Protein Fractionation Kit? If the protein concentration of fractions is not sufficient for your downstream applications, we recommend using 80% ice-cold acetone to concentrate the proteins. The resulting protein pellet can be resuspended in a buffer suitable for your downstream applications. Alternatively, protein concentration can be performed using appropriate spin devices containing low molecular weight cutoff membranes. How do I prepare the Cellular Protein Fractionation Kit fractions for 2D gel electrophoresis? Cytosol, Membrane/Organelle, and Nuclear proteins can be used directly for 2-D gel electrophoresis. Samples must be diluted 1: 4 with a common loading buffer for IEF (e.g., 5 M urea, 2 M thiourea, 4% CHAPS, ampholytes, 100 mM DTT) and incubated for 60 minutes at room temperature prior to loading on an IEF gel. Cytoskeleton proteins must include a clean-up step (e.g, ice-cold acetone precipitation, as outlined above), prior to loading the proteins on an IEF gel. For improved results in 2-D gel electrophoresis we strongly recommend precipitation (clean-up) of all 4 fractions prior to IEF.

IX. SAFETY CONSIDERATIONS

MSDS available upon request.

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X. REFERENCES

Zhang, L., and Insel, P. A. 2004. J. Biol. Chem. 279, 20858. Yuan, X., et al. 2002. Electrophoresis 23, 1185. Butcher, et al. 2001. J. Immunol. 167, 2193. Ott, et al. 2001. Pharmacogenomics J. 1, 142. Allen, L. 2000. Nature 405, 819. Dunn, M. J. 2000. Electrophoresis 6. Rabilloud, T. 2000. Biochem. Biophys. Res. Comm. 254, 93. Reymond, et al. 1997. Electrophoresis 18, 2842. Laemmli, U. K. 1970. Nature 227, 680. Lowry, et al. 1951. J. Biol. Chem. 193, 265. Sabio, G., et al. 2005. E M B O J . in press. Efanov, A.M., et al. 2004. D i a b e t e s 53, s75. Singh, L.P., et al. 2004. A m . J . P h y s i o l . R e n a l P h y s i o l . 286, F409.

XI. LICENSING

PerkinElmer® is a registered trademark of PerkinElmer Life and Analytical Sciences, Inc. EZQ® is a registered trademark of Invitrogen Corp. The technology contained herein is subject to a patent application owned by Merck KGaA.

XII. NAME AND PLACE OF MANUFACTURE

For further technical information or to place an order, call: PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA Shelton, CT 06484-4794 USA Phone: (800) 762-4000 or (+1) 203-925-4602

www.perkinelmer.com

[email protected]

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Europe: PerkinElmer Life & Analytical Sciences, Inc. Imperiastraat 8 B-1930 Zaventem Belgium +32 2 717 7911 [email protected] Outside of the U.S. and Europe: contact your local distributor Website: www.perkinelmer.com

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PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA Shelton, CT 06484-4794 USA Phone: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices ©2007 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. (PKI product names) are trademarks and (PKI product names) are registered trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries that are depicted herein are the property of their respective owners. PerkinElmer reserves the right to change this document at any time without notice and disclaims liability for editorial, pictorial or typographical errors.

PC5683-0507

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