Labeling Lumio™ Fusion Proteins, continued
Contents
1. The concentration of Disperse Blue 3 is qualified by spectrophotometry To ensure performance Disperse Blue 3 is also functionally tested using GripTite 293 MSR cells transfected with the pe DNA 6 2 nLumio GW p64 vector and using the protocols included in this manual Overview Introduction Advantages of the Lumio Labeling System Purpose of this Manual Introduction The Lumio In Cell Labeling Kits use the Lumio Technology to facilitate site specific fluorescent labeling of proteins in live mammalian cells Use of the Lumio Technology provides a sensitive method for in vivo detection and subcellular localization of proteins fused to the Lumio tag using fluorescence microscopy TM TM Using the Lumio Technology and the Lumio In Cell Labeling Kits for fluorescent labeling of recombinant proteins provides the following advantages TM e Small size of the Lumio tag 6 amino acids 585 Da is less likely to interfere with the structure or biological activity of the protein of interest e Lumio Labeling Reagents are membrane permeable and readily cross the cell membrane allowing labeling and detection of recombinant proteins in live mammalian cells TM TM e Lumio Labeling Reagents bind the Lumio tag with high specificity and high affinity nanomolar or lower dissociation constant allowing targeted labeling of the protein of interest TM e Lumio Labeling Reagents2. TM e Store Lumio Labeling Reagents at 20 C protected from light e Aliquot Lumio Labeling Reagents to avoid multiple freeze thaw cycles e Use freshly prepared labeling solution to label proteins Concentration of Lumio Reagent in labeling solution is too low TM Increase the concentration of Lumio Reagent in the labeling solution up to 10 uM Labeling time too short Increase the length of time cells are exposed to the Lumio Reagent Incorrect filter set used e Usea standard FITC filter set to detect proteins labeled with Lumio Green e Usea standard Texas Red filter set to detect proteins labeled with Lumio Red e Enhance visualization of proteins that are difficult to detect by using optimized filter sets see page 18 TM Lumio Red Labeling Reagent is photobleaching e Analyze fluorescent signal for only a few seconds at a time e Use a lower magnification objective e Decrease the lamp intensity continued on next page 21 Troubleshooting continued Problem Reason Solution High background fluorescence levels Too much serum in labeling media If you are using a cell type that tolerates overnight growth in reduced serum media culture the cells in Opti MEM or another reduced serum media overnight 16 18 hours and then repeat the labeling procedure Dilute the Lumio Reagent in HBSS and perform the wash s
3. Hank s Balanced Salt Solution HBSS and HEPES Buffer Saline HBS are suitable If you are culturing adherent cells make sure the labeling media contains calcium and magnesium to promote cell attachment For transfected cells we recommend labeling in medium containing 2 5 uM Lumio Labeling Reagent as a starting point For cells transduced with lentivirus a Lumio Reagent concentration of 1 25 uM may be optimal Depending on the levels of specific and background fluorescent signal you can optimize the Lumio Reagent concentration to better visualize your labeled protein We recommend trying a concentration range of 1 uM to 10 uM Lumio Reagent As a starting point we recommend labeling cells for 15 30 minutes Generally fluorescent signal is detectable 15 minutes after labeling and increases steadily for about 90 minutes We generally do not see any increase in the intensity of the fluorescent signal after 90 minutes Depending on the stability of your protein fluorescent signal may be visible up to 48 hours after labeling TM If you are labeling proteins with the Lumio Reagent for the first time we recommend optimizing the labeling time for your protein and cell line You can accomplish this by visualizing protein labeling every 15 minutes for up to 90 minutes using a fluorescence microscope see Detecting Lumio Fusion Proteins page 18 As fluorescent signal from the bound Lumio Reagent increases nonsp
4. Lumio Reagent before visualizing cells proceed to Performing Dual Labeling next page continued on next page 13 Labeling Lumio Fusion Proteins continued Protocol for Follow this protocol to label transfected cells in suspension with Lumio Green or Transfected Cells Lumio Red labeling solution We recommend including a positive expression in Suspension control and a non Lumio vector control in your experiment to determine foreground and background fluorescence see page 7 1 For each sample pellet cells by centrifugation and wash the cell pellet once with Opti MEM I Reduced Serum Medium 2 Resuspend the pellet with 1X Lumio labeling solution prepared with Opti MEM Medium see Preparing the Lumio Labeling Solution previous page to a final density of 1 x 10 cells ml Transfer the cells to your tissue culture format of choice Important Appropriately discard any unused Lumio labeling solution according to your institution s guidelines Do not reuse the 1X Lumio labeling solution 3 Cover the plate to prevent the solution from evaporating Incubate the cells at room temperature for 30 minutes protected from light Note Extending the incubation time may increase the fluorescent signal but may also increase the background 5 Ifyou are ready to detect your labeled protein using fluorescence microscopy proceed to Using Disperse Blue 3 next page If you are performing dual labeling and
5. use optimized filter sets with specifications that TM more closely match the excitation and emission maxima of the Lumio Reagents If desired you may use a color camera that is compatible with the microscope to photograph the cells We recommend using a digital camera or high sensitivity film such as 400 ASA or greater For optimal pictures we recommend photographing cells using a short exposure time 0 5 seconds or less although this time may vary depending on your protein and application Note If you are dual labeling your protein with the Lumio Green and Lumio Red Labeling Reagents you will need to capture the images separately and superimpose them using imaging or graphics software As with other fluorescent dyes avoid photo bleaching the labeled cells The Lumio Red Labeling Reagent is particularly sensitive to continuous illumination through a high magnification high numerical aperture objective with UV or any other wavelength of light that can excite the reagent To reduce photo bleaching limit exposure of cells to excitation light by analyzing fluorescent signal for a few seconds at a time Alternatively use a lower magnification objective or decrease the lamp intensity to reduce exposure of the substrate to light continued on next page Detecting Lumio Fusion Proteins continued What You Should Recombinant proteins fused to the Lumio tag will appear brightly labeled and See will emit a g
6. weights and micrograms supplied for each of the Labeling Lumio Labeling Reagent Reagents Reagent Molecular Weight pg Supplied Lumio Green Labeling Reagent 664 50 g mol 53 2 Lumio Red Labeling Reagent 545 38 g mol 43 6 vi Accessory Products Accessory Products TM Additional products that may be used with the Lumio In Cell Labeling Kits are available from Invitrogen For more information about these products refer to our Web site www invitrogen com or contact Technical Service page 23 Item Amount Catalog no Opti MEM I Reduced Serum Medium 1X 100 ml 31985 062 Liquid 500 ml 31985 070 Hank s Balanced Salt Solution HBSS with 500 ml 14025 092 calcium and magnesium but no phenol red IL 14025 076 HEPES Buffer Solution 1 M 20 ml 15630 106 100 ml 15630 080 vii Product Qualification Introduction Lumio Labeling Reagents Disperse Blue 3 viii TM The components of the Lumio In Cell Labeling Kits are qualified as described below TM TM The Lumio Green and Lumio Red Labeling Reagents are qualified as follows Purity Determined by HPLC Mass Determined by mass spectroscopy Functionality To ensure performance the Lumio Labeling Reagents are functionally tested using GripTite 293 MSR cells transfected with the pcDNA 6 2 nLumio GW p64 vector and using the protocols included in this manual
7. wish to label your protein with the second Lumio Reagent before visualizing cells proceed to Performing Dual Labeling below Protocol for Cells Follow this protocol to label cells transduced with lentivirus with Lumio Green Transduced with labeling solution You may plate your cells in any tissue culture format of choice Lentivirus We recommend that your cells are 60 90 confluent at the time of labeling for optimal results We also recommend including a positive expression control and a negative transduction control i e cells not treated with virus in your experiment to determine foreground and background fluorescence see page 7 1 Remove the growth medium from the cells and wash cells 2 3 times with HBSS 2 Add the appropriate amount of 0 5X Lumio labeling solution prepared with HBSS to each well see Preparing the Lumio Labeling Solution previous page Important Appropriately discard any unused Lumio labeling solution according to your institution s guidelines Do not reuse 0 5X Lumio labeling solution 3 Cover the plate to prevent the solution from evaporating Incubate the cells at room temperature for 15 minutes protected from light Note Extending the incubation time may increase the fluorescent signal but may also increase the background 5 To detect your labeled protein using fluorescence microscopy proceed to Using Disperse Blue 3 page 16 If you are performing dual labeling and wish to label yo
8. 11 Labeling Lumio Fusion Proteins continued Recommended Use the following recommended conditions to label your protein with the Labeling Lumio Reagent For more information see the section entitled General Conditions Guidelines to Use the Lumio Labeling Kits page 6 Condition Recommendation Tissue culture format e You may plate cells in any size tissue culture plate of choice e g 6 well format e Make sure that your tissue culture plate is compatible with your detection instrument Cell density For optimal labeling efficiency e Plate adherent cells such that they will be 60 90 confluent at the time of labeling e Label suspension cells at a density of 1 2 x 10 cells ml Labeling Media For optimal efficiency label cells in Opti MEM Reduced Serum Medium HBSS or HBS Note If you are culturing adherent cells make sure the labeling media contains calcium and magnesium to promote cell attachment Concentration of Lumio e For transfected cells incubate in 2 5 uM TM Labeling Reagent Lumio Reagent e For cells transduced with lentivirus incubate in 1 25 uM Lumio Reagent To optimize fluorescent signal vary the Lumio Reagent concentration from 1 uM to 10 uM Labeling time For most applications label proteins for 15 30 minutes Materials Needed Be sure to have the following materials on hand before beginning e Lumio Labeling Reagent supplied wit
9. USA by applying a full vial of material to the mouse skin In this study no adverse reaction or toxicity was noted Although arsenic compounds are toxic this product contains lt 0 2 of an organic arsenic compound that shows no toxicity at a maximum dose level likely to be handled The toxicology of this material however has not been fully investigated Handle according to your chemical hygiene plan and prevent contact with this material TM Treat accidental spills of the Lumio Labeling Reagents on surfaces with 10 bleach for 10 minutes and then carefully clean up Discard arsenic containing waste according to your institution s guidelines TM Treat accidental contact of the Lumio Labeling Reagents with human skin by washing excess reagent off with soap and water as soon as possible Do not treat arsenic skin exposure with EDT 1 2 ethanedithiol since EDT may promote uptake of arsenic reagents into the body Consult a physician following contact Methods General Guidelines for Lumio Labeling Introduction Factors to Consider When Choosing a Labeling Reagent Phenotypic Effects With Lumio Red TM The Lumio In Cell Labeling Kits facilitate site specific labeling and detection of recombinant proteins in live mammalian cells Using the kit allows you to monitor cellular protein expression under real time physiological conditions Once recombinant proteins are labeled and detected using fluorescence m
10. als 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 to not 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 researc
11. arization products available from Invitrogen visit our Web site www invitrogen com or contact Technical Service page 23 TM If you label your protein with the Lumio Red Reagent you may detect the labeled protein using electron microscopy The Lumio Red Labeling Reagent photoconverts diaminobenzidine into a highly localized precipitate that can be stained and detected under an electron microscope Adams et al 2002 Gaietta et al 2002 The Lumio Green Reagent does not catalyze this photoconversion process and thus is not compatible with electron microscopy Labeling Lumio Fusion Proteins Introduction Important Note Thawing and Aliquoting Lumio Reagent TM To label recombinant proteins fused to the Lumio tag you will need to incubate your cells with Lumio Labeling Reagent Guidelines and instructions are provided in this section to label your recombinant protein We recommend waiting a minimum of 24 hours post transfection or 48 hours post transduction before labeling to ensure adequate expression of your protein If you plan to culture cells further after labeling with Lumio Reagent be sure to maintain sterility throughout the experiment e Perform all manipulations within a tissue culture hood e Prepare solutions using sterile reagents Previously published articles regarding FIAsH labeling of proteins required addition of 1 2 ethanedithiol EDT during the labeling proce
12. become strongly fluorescent only upon binding the Lumio tag allowing specific detection of Lumio tagged proteins This manual provides the following information e An overview of the Lumio Technology including the Lumio Green and Lumio Red Labeling Reagents TM e Guidelines and instructions for using the Lumio Labeling Reagents to label your protein of interest e Guidelines and instructions to detect your Lumio labeled protein in live mammalian cells using fluorescence microscopy Lumio Technology Introduction Components of the Lumio System Tetracysteine Motif Lumio Green Labeling Reagent TM Lumio In Cell Labeling Kits are based on FIAsH Fluorescein Arsenical Hairpin technology which uses biarsenical labeling reagents to bind and detect proteins containing a tetracysteine motif Griffin et al 1998 The biarsenical labeling reagents are nonfluorescent until they bind the tetracysteine motif at which time they become highly fluorescent The Lumio System consists of two major components TM e The tetracysteine Lumio tag Cys Cys Pro Gly Cys Cys When fused to a gene of interest in the context of a Lumio vector the Lumio tag allows the expressed fusion protein to be specifically recognized by a biarsenical labeling reagent For more information on the tetracysteine motif see below e A biarsenical labeling reagent Lumio Green or Lumio Red whi
13. ch becomes fluorescent upon binding to recombinant proteins containing the Lumio tag The Lumio Green and Lumio Red Labeling Reagents are supplied pre complexed to the dithiol EDT 1 2 ethanedithiol which stabilizes and solubilizes the biarsenic reagents For information on how the Lumio Reagent binds the Lumio tag and becomes fluorescent see the next page The Lumio Reagents bind a tetracysteine motif consisting of Cys Cys Xaa Xaa Cys Cys where Cys equals cysteine and Xaa equals any amino acid other than cysteine This motif is rarely seen in naturally occurring proteins allowing specific fluorescent labeling of recombinant proteins fused to the Lumio tag In the Lumio System the optimized Cys Cys Pro Gly Cys Cys tetracysteine motif is used as this motif has been shown to have a higher affinity for and more rapid binding to biarsenic compounds as well as enhanced stability compared to other characterized motifs Adams et al 2002 The Lumio Green Labeling Reagent is based on the FIAsH reagent and is a nonfluorescent biarsenical derivative of fluorescein Griffin et al 1998 Lumio Green is supplied pre complexed to EDT is membrane permeable and readily enters the cell See the figure below for the structure of the Lumio Green Reagent Sa SS zS Formula C24H18sAS20 S4 A HO o S Molecular Weight 664 50 eee gt COOH continued on next page Lumio Technology continued Lumio Re
14. ct into the cell line of choice for transient or stable expression of the gene of interest Mammalian Lumio Gateway Vectors or e Deliver and express your construct in either dividing or non dividing mammalian cells via a replication incompetent HIV 1 based lentivirus ViraPower II Lentiviral Lumio Gateway Vectors The Lumio Technology works best for labeling proteins that are expressed at high levels or are concentrated in a subcellular region If you are expressing proteins at low levels e g from a weak promoter or if you are expressing a cytoplasmic protein we recommend conducting initial studies in transiently transfected cells where protein expression levels are higher or transducing cells at a higher MOI e g 10 100 e We recommend designing your experiment so that cells will be at optimal density at the time of labeling Suspension cells typically label most efficiently at a density of 1 2 x 10 cells ml Adherent cells label most efficiently when they are 60 90 confluent at the time of labeling e For cells transduced with lentivirus it may be useful to culture the cells in Opti MEM Reduced Serum Media or another reduced serum media overnight 16 18 hours before labeling to reduce the background from serum e For transfection experiments we recommend transfecting cells with a positive expression control and a non Lumio vector control i e a vector that does not encode the Lumio tag
15. d Example If you added 1 ml of Lumio labeling solution to each well in a 6 well plate you will need 12 ml of 1X Disperse Blue 3 solution 2 ml per well To make the 1X stock solution add 12 ul of Disperse Blue 3 to 12 ml of Opti MEM Medium 2 After the incubation with the Lumio labeling solution carefully remove the Lumio labeling solution and discard appropriately Wash cells once with Opti MEM Medium for transfected cells or HBSS for transduced cells and discard appropriately 3 Gently add the appropriate amount of 1X Disperse Blue 3 solution to each well Do not remove the solution You will visualize your labeled protein in the presence of the Disperse Blue 3 solution Proceed to Detecting Lumio Fusion Proteins next page continued on next page Using Disperse Blue 3 continued Purchasing Disperse Blue 3 You may purchase additional Disperse Blue 3 in powder form from Fisher Scientific Catalog no AC20158 0500 Follow the instructions below to prepare a 20 mM Disperse Blue 3 solution 1 Add 5 9 mg of Disperse Blue 3 powder Fisher Catalog no AC20158 0500 to 1 ml of anhydrous DMSO Dissolve powder by vortexing Filter sterilize the solution through a 0 2 um nylon filter Aliquot the solution into multiple tubes Store at 20 C 17 Detecting Lumio Fusion Proteins Introduction Recommended Filter Sets Note Color Camera 18 After you have labeled your protein yo
16. d Labeling Reagent How the Lumio Reagents Bind the Lumio Tag The Lumio Red Labeling Reagent is based on the ReAsH reagent and is a nonfluorescent biarsenical derivative of the red fluorophore resorufin Gaietta et al 2002 Lumio Red is supplied pre complexed to EDT is membrane permeable and readily enters the cell See the figure below for the structure of Lumio Red Reagent Formula Cy6Hi3As2NO3S4 CR S ij Molecular Weight 545 38 ZA N Both the Lumio Green and Lumio Red biarsenic labeling reagents bind the Lumio tag through four covalent bond formations the two arsenic groups of the Lumio Reagents each bind two thiols in the Lumio tag tetracysteine sequence see diagram below Upon binding the Lumio Reagents are converted to a highly fluorescent state which can be detected at the appropriate emissions peak see next page Protein of Interest Cys Cys Pro Gly Cys Cys G Lumio Tag xO x i Lumio Green Labeling Reagent Lumio Green Labeling Reagent non fluorescent fluorescent continued on next page Lumio Technology continued Fluorescence Spectra for the Lumio Reagents Once the Lumio Labeling Reagent binds the Lumio tag fused to your protein the reagent will emit a green Lumio Green or red Lumio Red fluorescent signal when excited at the appropriate wavelength The fluorescent excitation and TM TM emission spectra for th
17. dure and during subsequent washing steps to reduce nonspecific binding Adams et al 2002 Griffin et al 2000 Griffin et al 1998 Due to the odor and toxicity of EDT however the labeling procedures provided in this section have been specifically developed to not require addition of EDT Although EDT is not required for the labeling procedure you may add EDT to help you visualize proteins that are expressed at low levels e g diffuse cytoplasmic proteins or if you detect unusually high background We recommend using an EDT concentration that is four to ten times greater than the concentration of Lumio Reagent in the labeling media EDT Sigma Aldrich Catalog no W34 840 6 has a strong odor and must be handled in a fume hood Refer to the Material Safety Data Sheet before handling We recommend aliquoting the Lumio Labeling Reagent to minimize freeze thaw cycles Let the frozen Lumio Reagent warm to room temperature protected from light and aliquot 5 10 ul into multiple tubes When using individual aliquots warm to room temperature protected from light and remove the desired amount of reagent Immediately recap the tube to reduce moisture uptake Store aliquots at 20 C protected from light Note Both the Lumio Green and the Lumio Red Reagents may change color during storage due to changes in pH This color change is normal and does not affect the performance of the reagent continued on next page
18. e Lumio Green and Lumio Red Labeling Reagents are provided below 100 4 A Lumio Green DA 2 Excitation gt 2 80 J i i 2 I u 60 J Lumio Green E Emission Q O 5 404 i L a a f Pi yt Lumio Red s Emission Excitation Lumio Red 400 450 500 550 600 Wavelength nm 650 700 750 Labeling Reagent Excitation Maximum Emission Maximum Lumio Green 508 nm 528 nm Lumio Red 593 nm 608 nm Working with Arsenic Compounds Introduction Dermal Toxicity Evaluation Accidental Spills and Accidental Contact TM TM The Lumio Green and Lumio Red Labeling Reagents are biarsenical compounds and should be handled with care See below for general guidelines Exercise caution when handling the Lumio Green and Lumio Red Labeling Reagents We recommend the following guidelines e Review the Material Safety Data Sheet before handling e Wear protective clothing eyewear and gloves suitable for use with dimethyl sulfoxide e g nitrile gloves when handling the Lumio Labeling Reagents e Discard all excess reagents that contain or have come in contact with arsenic according to your institution s guidelines and all applicable local state and federal requirements TM A dermal toxicity evaluation of the Lumio Labeling Reagents was independently performed by MB Research Laboratories Spinnerstown PA
19. e background fluorescent signal Adams et al 2002 Follow the guidelines below to prepare and add Disperse Blue 3 solution to your cells Be sure to have the following materials on hand before beginning e Disperse Blue 3 supplied with the Lumio In Cell Labeling Kit warm to room temperature e Opti MEM Reduced Serum Medium Catalog no 31985 062 e HBSS with calcium and magnesium for lentivirus transduced cells Catalog no 10425 092 e Mammalian cell line of interest incubating with Lumio labeling solution We recommend storing multiple aliquots of Disperse Blue 3 at 20 C to minimize freeze thaw cycles Store tubes in use at 4 C Disperse Blue 3 may cause eye and skin irritation Wear protecting clothing eyewear and gloves when handling Refer to the Material Safety Data Sheet before handling Disperse Blue 3 is supplied as a 1000X solution Make the 1X stock solution just before use and keep at room temperature until needed Follow the instructions below to prepare a 1X stock solution of Disperse Blue 3 and to add it to your cells 1 To make a 1X stock solution add the appropriate amount of Disperse Blue 3 to Opti MEM Medium for transfected cells or HBSS for transduced cells Vortex to mix 1X solution is 20 uM Disperse Blue 3 You will add twice the volume of Disperse Blue 3 solution to each well as you did the Lumio labeling solution see table on page 13 Keep at room temperature until neede
20. ecific background fluorescence will also increase Visualizing your cells every 15 minutes will allow you to determine the labeling time that gives you the optimal signal to noise ratio A General Guidelines for Dual Lumio Labeling Introduction Note Which Lumio Reagent to Use First When to Use the Second Lumio Reagent TM The Lumio Dual Labeling Kit allows successive labeling of the protein of interest in living cells in order to distinguish between older and newly made protein molecules Using such pulse chase assays to temporally distinguish pools of protein aid in the study of protein assembly protein internalization and protein turnover Gaietta et al 2002 In addition to the guidelines provided in the previous section refer to the following guidelines when using the Lumio Green and Lumio Red Reagents for dual labeling assays TM Both the Lumio Green and the Lumio Red Reagents bind the same tetracysteine motif i e Lumio tag and therefore can only be used to label one recombinant protein at any given time Do not use the Lumio Green and Lumio Red Reagents to separately label two different proteins If you wish to detect two proteins in the same cell you will need to use two separate reporter systems Example To detect two different proteins in the same cell express one protein fused to the Lumio tag and label with Lumio Red Labeling Reagent Express a second prot
21. ein fused to GFP Detect both proteins using fluorescence microscopy and the appropriate filter sets To dual label your protein of interest you will pulse label your protein with the first Lumio Reagent followed by a chase label with the second Lumio Reagent You will follow the same labeling procedure for both Lumio Reagents see pages 13 14 Because the Lumio Red Reagent can cause transient phenotypic effects on cells see page 6 we generally recommend using Lumio Green as the first labeling reagent TM After the first labeling procedure with Lumio Green is performed you may do any of the following e Immediately label the protein with the second reagent Lumio Red e Incubate cells for desired length of time up to 48 hours depending on the stability of your protein before labeling with Lumio Red e Visualize protein labeled with Lumio Green under a fluorescence TM microscope before labeling with Lumio Red General Guidelines for Detecting Lumio Fusion Proteins Introduction Note Fluorescence Polarization Electron Microscopy 10 We recommend using fluorescence microscopy to detect and localize fluorescently labeled proteins Guidelines for performing fluorescence microscopy are provided in the section entitled Detecting Lumio Fusion Proteins see page 18 You may also detect fluorescent signal using fluorescence polarization or if your protein is lab
22. eled with Lumio Red electron microscopy see below Although the Lumio In Cell Labeling Kits are primarily designed for protein detection and localization using fluorescence microscopy it is possible to quantitatively analyze fluorescent signal using a fluorescence plate reader or perform fluorescence activated cell sorting FACS using a flow cytometer However depending on your protein expression levels and your application background noise may be too high to accurately obtain and quantify fluorescent signal readings using these methods If you will be analyzing fluorescent signal using a fluorescence plate reader or a flow cytometer consider the following e Make sure the instrument is equipped with the proper optical filters to detect Lumio Green or Lumio Red fluorescent signal e Include the proper negative controls in your experiment to determine background fluorescence levels Fluorescence polarization assays provide information regarding molecular orientation and mobility and are used to study multiple processes including receptor ligand interactions protein degradation and membrane fluidity In contrast to conventional fluorescent dyes which attach through rotationally mobile single bonds the Lumio Labeling Reagents bind the Lumio tag through four rigid covalent bond formations making the Lumio System ideal for fluorescence polarization assays Adams et al 2002 For information on fluorescence pol
23. er Well 6 well 1ml 12 well 500 ul 24 well 250 ul 48 well 100 ul 96 well 50 pl Protocol for Follow this protocol to label transfected adherent cells with Lumio Green or Transfected Lumio Red labeling solution You may plate your cells in any tissue culture Adherent Cells format of choice We recommend that your cells are 60 90 confluent at the time of labeling for optimal results We also recommend including a positive expression control and a non Lumio vector control in your experiment to determine foreground and background fluorescence see page 7 1 Remove the growth medium from the cells and wash cells once with Opti MEM I Reduced Serum Medium 2 Add the appropriate amount of 1X Lumio labeling solution prepared with Opti MEM Medium to each well see Preparing the Lumio Labeling Solution above Important Appropriately discard any unused Lumio labeling solution according to your institution s guidelines Do not reuse the 1X Lumio labeling solution 3 Cover the plate to prevent the solution from evaporating Incubate the cells at room temperature for 30 minutes protected from light Note Extending the incubation time may increase the fluorescent signal but may also increase the background 5 If you are ready to detect your labeled protein using fluorescence microscopy proceed to Using Disperse Blue 3 page 16 If you are performing dual labeling and wish to label your protein with the second
24. h contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 References Adams S R Campbell R E Gross L A Martin B R Walkup G K Yao Y Llopis J and Tsien R Y 2002 New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo Synthesis and Biological Applications J Am Chem Soc 124 6063 6076 Gaietta G Deerinck T J Adams S R Bouwer J Tour O Lair D W Sosinsky G E Tsien R Y and Ellisman M H 2002 Multicolor and Electron Microscopic Imaging of Connexin Trafficking Science 296 503 507 Griffin B A Adams S R Jones J and Tsien R Y 2000 Fluorescent Labeling of Recombinant Proteins in Living Cells with FIAsH Meth Enzymol 327 565 578 Griffin B A Adams S R and Tsien R Y 1998 Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells Science 281 269 272 2003 2005 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 25 8 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
25. h the Lumio In Cell Labeling Kit warm to room temperature protected from light e Opti MEM Reduced Serum Medium Catalog no 31985 062 e HBSS with calcium and magnesium for lentivirus transduced cells Catalog no 10425 092 e Mammalian cell line of interest expressing Lumio tagged recombinant protein plated in the tissue culture format of choice or in suspension as appropriate continued on next page 12 Labeling Lumio Fusion Proteins continued TM TM Preparing the The Lumio Green and Lumio Red Labeling Reagents are provided as an 800X Lumio Labeling solution Follow the guidelines below to make a 1X labeling solution Solution e For transfected cells add the appropriate amount of Lumio Reagent to Opti MEM Medium to make a 1X labeling solution and vortex to mix 1X labeling solution is 2 5 uM Lumio Reagent Make just enough 1X labeling solution for your immediate needs e For lentivirus transduced cells add the appropriate amount of Lumio Reagent to HBSS to make a 0 5X labeling solution and vortex to mix 0 5X labeling solution is 1 25 uM Lumio Reagent Make just enough 0 5X labeling solution for your immediate needs e Make the labeling solution just before use and keep at room temperature until needed e Refer to the table below for recommended amounts of labeling solution to use for various tissue culture formats Culture Vessel Labeling Solution p
26. ications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 23 Purchaser Notification Introduction Limited Use Label License No 167 Target Sequences for Synthetic Molecules 24 TM Use of the Lumio In Cell Labeling Kits is covered under the license detailed below This product and or its use is the subject of one or more of U S Patent Nos 5 932 474 6 008 378 6 054 271 and 6 451 569 and foreign equivalents owned by and or licensed to Invitrogen Corporation 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 materi
27. icroscopy cells may be cultured further for use in additional assays or other downstream applications Invitrogen offers the Lumio Green Lumio Red and Lumio Dual Green and Red In Cell Labeling Kits for fluorescent labeling of proteins fused to the Lumio tag Because we have noticed transient phenotypic effects using the Lumio Red Labeling Reagent see below and because the Lumio Red Reagent is sensitive to photo bleaching when exposed to continuous illumination we generally recommend using the Lumio Green Labeling Kit to label your recombinant protein TM The Lumio Red Labeling Kit offers an alternative labeling strategy that allows you to perform additional applications We recommend using the Lumio Red Labeling Kit for the following conditions e You wish to perform dual labeling experiments on the same Lumio tagged protein using both the Lumio Green and Lumio Red Labeling Reagents e You are already using a green fluorescent reporter e g GFP FITC conjugated antibody in your assays and require a second labeling reagent e You wish to detect labeled proteins by electron microscopy see page 10 for more information We have noticed changes in cell morphology approximately 24 hours after labeling proteins with Lumio Red Reagent in HEK293 and COS 7 cell lines Cells appear to round up and the general shape and pattern of the cells appear disrupted This phenotypic effect is transient as cel
28. invitrogen Lumio In Cell Labeling Kits For site specific fluorescent labeling and detection of Lumio tagged proteins in live mammalian cells Catalog nos 12589 040 and 12589 057 Version C 27 June 2005 25 0700 ii Table of Contents Kit Contents ADO Storag ennegar ieten Liso habas seed a ebanista bi e ER v JACCESSOLY POL TE vii Product Qualification EE viii Introduction uuuzannsannnonnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnsnnnsnnnnannnannnann 1 EENEG 1 Lumio Ke airline 2 Working with Arsenic Compound Ssstt eaen Ea aE EESE a Eii e EERE TEE ARESE Eea EESE TET 5 lettre Ee 6 General Guidelines for Lumio Labeling ti een as 6 General Guidelines tor Dual Lumio Labeling u ci aa NA Rie as 9 General Guidelines for Detecting Lumio Fusion Proteins is conciomsicciancia orion ois ecards cbc 10 Labeling Lumio Fusion Protesis az 11 Using Disperse blue Sata rd na 16 Detecting Lumio EE 18 No 41219 2 EE 21 Troubleshooting a HE Id Li Ae 21 Ee EE 23 Purchaser Notification et eege Eed de Paai oaken daea aask Eae 24 References nda E ea odo adan 25 iii iv Kit Contents and Storage Types of Kits This manual is supplied with the products listed below Product Catalog no Lumio Red In Cell Labeling Kit 12589 040 Lumio Green In Cell Labeling Kit 12589 057 Additional Kits The following products include one or both of the kits l
29. io Red Reagent showing transient phenotypic effects Phenotypic effects are transient and should disappear after approximately 48 hours see page 6 Photographs of cells show high background fluorescence Exposure time too long Take photographs using a short exposure time 0 5 seconds or less 22 Technical Service Web Resources Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service contact information click on Contact Us e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 35730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_service invitrogen com E mail jpinfo invitrogen com E mail eurotech in
30. isted above Product Catalog no Mammalian Lumio Gateway Vectors with Lumio 12589 016 Green In Cell Labeling Kit Mammalian Lumio Gateway Vectors with Lumio Red 12589 024 In Cell Labeling Kit Mammalian Lumio Gateway Vectors with Lumio 12589 032 Dual Green and Red In Cell Labeling Kit ViraPower II Lentiviral C Lumio Gateway Expression K370 20 System ViraPower II Lentiviral N Lumio Gateway Expression K371 20 System Shipping Storage The Lumio In Cell Labeling Kits are shipped on blue ice Upon receipt store as detailed below Kit Storage Lumio Red In Cell Labeling Kit TM Lumio Red 20 C protected from light Disperse Blue 3 20 C Lumio Green In Cell Labeling Kit Lumio Green 20 C protected from light Disperse Blue 3 20 C TM Lumio In Cell The following reagents are supplied with the Lumio In Cell Labeling Kits Store TM Labeling Reagents the Lumio Green or Lumio Red Labeling Reagent at 20 C protected from light Store Disperse Blue 3 at 20 C All components are stable for at least 6 months under these conditions Reagent Composition Amount In Cell Labeling Reagent 2 mM in DMSO 40 pl Lumio Green or Lumio Red Disperse Blue 3 20 mM in DMSO 200 pl continued on next page Kit Contents and Storage continued Molecular Weights The table below lists the molecular
31. ls recover 48 hours after labeling and is thought to be due to the generation of singlet oxygens when the Lumio Red Reagent is exposed to high intensity light Adams et al 2002 We have not observed this effect on cells labeled with the Lumio Green Labeling Reagent TM If you are using the Lumio Red Reagent to label your protein you may observe similar morphological changes in your cells depending on your cell line expressed recombinant protein and application Note that these phenotypic effects may transiently influence any downstream assays you wish to perform continued on next page General Guidelines for Lumio Labeling continued Lumio Vectors Expressing Your Gene of Interest with the Lumio Tag Labeling Cells Controls TM Before you use one of the Lumio Labeling Kits to label and detect your protein of interest in vivo you must generate an expression construct containing your gene of interest fused to the Lumio tag The Mammalian Lumio Gateway Vectors and the ViraPower II Lentiviral Lumio Gateway Vectors are available from Invitrogen to generate N terminal or C terminal Lumio tag fusion proteins For more information about these vectors see the vector manual visit our Web site www invitrogen com or contact Technical Service page 23 Once you have generated an expression construct containing your gene fused to the Lumio tag you may e Transfect your constru
32. nce with Opti MEM Medium for transfected cells or HBSS for transduced cells and repeat the labeling procedure with the second Lumio labeling solution e If you will be incubating your cells between labeling procedures remove the Disperse Blue 3 solution and discard appropriately Wash cells once with Opti MEM Medium add complete growth medium to the cells and incubate at 37 C for the desired amount of time Repeat the labeling procedure with the TM second Lumio labeling solution 20 Appendix Troubleshooting This section lists potential problems and possible solutions that may help you troubleshoot your Lumio labeling experiments Introduction Problem Reason Solution Weak or no fluorescent signal Low expression of Lumio tagged protein e Increase protein labeling time e Increase concentration of Lumio Reagent in the labeling solution up to 10 uM e Culture cells for a longer period of time before labeling to ensure adequate protein expression e Re assess transfection conditions e For lentivirus transduced cells transduce cells at a higher MOI e g 10 100 Poor transfection efficiency e Re assess transfection conditions TM e Use Lipofectamine 2000 for transfection Poor transduction efficiency See the troubleshooting section of the ViraPower Lentiviral Expression System manual TM Lumio Reagents have lost activity
33. reen Lumio Green or a red Lumio Red fluorescent signal that should be easy to detect above the background fluorescence Note that cells will appear lightly and uniformly green or red depending on the Lumio Labeling Reagent used This is normal background fluorescence caused by autofluorescence of the cells autofluorescence of unbound Lumio Reagent and nonspecific binding of the Lumio Reagent For lentivirus transduced cells use the negative control mock transduced cells to determine the level of TM background Lumio fluorescence If you have trouble detecting your labeled protein above the background fluorescence see the recommendations below SEL If you are experiencing a high level of background fluorescence or if your Se 7 foreground fluorescent signal is low try the following o a Ei e Decrease the lamp intensity of the microscope e Adjust the concentration of the Lumio Reagent in the labeling solution and repeat the labeling procedure Use a range of 1 to 10 uM Lumio Reagent e If you used Opti MEM P to dilute the Lumio reagent and wash your cells use HBSS in place of Opti MEM as described in Protocol for Cells Transduced with Lentivirus on page 14 e Ifyou are using a cell type that tolerates overnight growth in reduced serum media culture the cells in Opti MEM or another reduced serum media overnight 16 18 hours and then repeat the labeling procedure e Adjust the inc
34. teps in the labeling procedure with HBSS as described in the procedures for lentivirus transduced cells Concentration of Lumio Reagent in labeling solution is too high Decrease the concentration of Lumio Reagent in the labeling solution to as low as 1 uM Labeling time too long Decrease the length of time cells are exposed to TM the Lumio Reagent Nonspecific binding of the Lumio Reagent Add EDT to the labeling media and to wash steps see page 11 for more information Add 20 uM Disperse Blue 3 solution to cells and visualize under the microscope Cells exhibit a green Lumio Reagent not e Remove Lumio Reagent and wash once or red hazy removed prior to visualizing with Opti MEM Medium background cells under the microscope e Add 20 uM Disperse Blue 3 solution to cells and visualize under the microscope Disperse Blue 3 solution not e Add 20 uM Disperse Blue 3 solution to cells added to cells and visualize under the microscope Cells detach from the Cells are disturbed when Gently add and remove reagents during surface of the well reagents are added and removed transfection labeling and wash steps Labeling media does not contain calcium or magnesium Use labeling media that contains calcium and magnesium to promote cell attachment e g Opti MEM I Reduced Serum Medium Altered cell morphology e g cells appear rounded up TM Cells exposed to Lum
35. to control for fluorescent artifacts or alterations in cell morphology that could affect the labeling and detection of your protein e For lentivirus transduction experiments we recommend using a positive expression control and a negative transduction control To prepare the negative control follow the transduction procedure in the ViraPower Lentiviral Expression System manual except omit the virus Then use the mock transduced cells in the labeling procedure to determine the level of background Lumio fluorescence continued on next page General Guidelines for Lumio Labeling continued Factors Affecting Protein Labeling Labeling Media Concentration of Lumio Reagent Labeling Time A number of factors can influence the degree of protein labeling and consequently the success of your detection experiment These factors include e Composition of labeling media e Concentration of Lumio Labeling Reagent used to label proteins e Length of incubation with Lumio Labeling Reagent Each of these factors is discussed further in this section TM Because the Lumio Reagent will nonspecifically bind to serum proteins including bovine serum albumin BSA we recommend using labeling media that is serum free or contains low amounts of serum 1 2 serum Opti MEM Reduced Serum Medium 1X is available separately from Invitrogen see page vii for ordering information Other buffered solutions including
36. u may detect protein expression and localization in live cells by visually observing the fluorescence of the bound Lumio Reagent General guidelines are provided below to select the type of microscope and filter sets to optimally visualize your labeled protein using fluorescence microscopy Fluorescent signal from Lumio Green or Lumio Red may be visible for up to 48 hours depending on the stability of your protein A standard fluorescein FITC filter set is suitable for visualizing proteins labeled with the Lumio Green Labeling Reagent A standard Texas Red filter set is suitable for visualizing proteins labeled with the Lumio Red Labeling Reagent Refer to the table below for the maximum excitation and emission values for each Lumio Reagent For a diagram of the fluorescence emission spectra for the TM Lumio Reagents see page 4 Labeling Excitation Emission Recommended Reagent Maximum Maximum Filter Set Lumio Green 508 nm 528 nm FITC Lumio Red 593 nm 608 nm Texas Red While the FITC and Texas Red filter sets are suitable for viewing proteins labeled with the Lumio Green and Lumio Red Labeling Reagents respectively the transmission wavelengths for these filter sets do not precisely overlap with the excitation and emission maxima of the Lumio Reagents If you experience difficulty detecting your protein due to low expression levels or unusually high background you may need to
37. ubation time of the cells with the Lumio labeling solution Visualize cells every 15 minutes up to 90 minutes to optimize the labeling time Detecting p64 If you transfected cells with the peDNA 6 2 nLumio GW p64 control plasmid supplied with the Mammalian Lumio Gateway Vectors you will detect p64 expression in the nucleoli p64 will appear as discreet brightly labeled punctate spots localized within the nuclei of cells Culturing Cells If you plan to culture cells further after detecting your labeled recombinant Further protein carefully remove the labeling solution from your cells and discard appropriately Wash cells once with Opti MEM Medium add complete growth medium and continue to culture cells at 37 C Reminder If you labeled your cells with Lumio Red Labeling Reagent you may notice transient phenotypic effects approximately 24 hours after performing the labeling procedure see page 6 continued on next page 19 Detecting Lumio Fusion Proteins continued Performing Dual If you are performing dual labeling and are ready to label your protein with the Labeling second Lumio Reagent we recommend you do the following TM e Prepare the second Lumio labeling solution right before use see Preparing the Lumio Labeling Solution page 13 e If you will be immediately labeling your protein with the second reagent remove the Disperse Blue 3 solution and discard appropriately Wash cells o
38. ur protein with the second Lumio Reagent before visualizing cells proceed to Performing Dual Labeling next page continued on next page 14 Labeling Lumio Fusion Proteins continued TM Performing Dual If you wish to label your protein with the second Lumio Reagent before Labeling visualizing cells under a microscope we recommend you do the following TM e Prepare the second Lumio labeling solution immediately before use see Preparing the Lumio Labeling Solution page 13 e Ifyou will be immediately labeling your protein with the second reagent remove the Lumio labeling solution and discard appropriately Wash cells once with Opti MEM Medium for transfected cells or HBSS for transduced cells and repeat the labeling procedure with the second Lumio labeling solution e If you will be incubating your cells between labeling procedures remove the first Lumio labeling solution and discard appropriately Wash cells once with Opti MEM Medium add complete growth medium to the cells and incubate at 37 C for the desired amount of time Repeat the labeling TM procedure with the second Lumio labeling solution 15 Using Disperse Blue 3 Introduction Materials Needed Aliquoting Disperse Blue 3 Preparing and Adding the Disperse Blue 3 Solution 16 Disperse Blue 3 is a nonfluorescent hydrophobic dye that is supplied with the Lumio In Cell Labeling Kits to reduce th
39. vitrogen com Material Data Safety Sheets MSDSs Limited Warranty MSDSs are available on our Web site at www invitrogen com On the home page click on Technical Resources and follow instructions on the page to download the MSDS for your product Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publ
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