CLIP-tag Cell Surface Trial Kit

Transcription

1 CLIP-tag Cell Surface Trial Kit for evaluating specific cell-surface CLIP-tag labeling with novel cell lines and imaging technologies Product o. LK347 Introduction This kit enables users to rapidly evaluate selective cellsurface labeling using the CLIP-tag technology with their cell lines and microscopy equipment. This kit contains the reagents required to selectively label cell-surface exposed CLIP-tag fusion proteins in living cells with non-cell permeable green (CT-488) and red (CT-547) fluorescent CLIP-tag substrates. CT-488 is based on the ATTO-TEC dye ATTO-488, and is suitable for standard fluorescein filter sets. It has an excitation maximum at 506 nm and an emission maximum at 526 nm. CT-547 is based on the Dyomics dye DY-547, and is suitable for standard TAMRA or Cy3 filter sets. It has an excitation maximum at 554 nm and an emission maximum at 568 nm. The kit also contains a localization control plasmid, pcems1-clip10m-k1r, for the transient or stable expression of the tagged eurokinin-1 receptor (K1R) in mammalian cells. The CLIP K1 receptor expressed from this plasmid localizes to the cell surface with the tag exposed to the extracellular side of the plasma membrane. This kit includes one vial of CT-488 and one vial of CT-547, each sufficient to make 4 ml of labeling medium. CLIP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. CLIP-tag is a small protein tag based on mammalian O6-alkylguanine-DA-alkyltransferase (AGT). CLIP-tag substrates are derivatives of benzylcytosine (CT). In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the reactive cysteine of CLIP-tag forming a stable thioether link. Although CLIP-tag is based on the same protein as SAP-tag, the benzylcytosine substrates form a separate class of substrates, different from the benzylguanine substrates recognized by SAP-tag. CLIP-tag and SAP-tag can be used for orthogonal simultaneous labeling. There are two steps to the use of this system: sub-cloning and expression of the protein of interest as a CLIP-tag fusion, and labeling of the fusion with the CLIP-tag substrate of choice. Expression of CLIP-tag fusion proteins is described in the documentation supplied with CLIP-tag plasmids. Cloning and expression of CLIP-tag fusion proteins are described in detail in the instructions supplied with CLIP-tag plasmids. Transient expression and labeling of CLIP-tag fusion proteins with CLIP-tag substrates are described in this document. The structure of the dye ATTO 488 is the undisclosed property of Atto-Tec, Siegen, Germany ( For further information please contact Atto-Tec directly Wavelength [nm] Figure 1. Excitation (dotted line) and emission spectra of label 488 coupled to SAP-tag / CLIP-tag in buffer at ph O ao 3 S H O H 2 O 3 S Figure 2. Structure of CT-547 (MW ) Wavelength [nm] Figure 3. Excitation (dotted line) and emission spectra of label 547 coupled to SAP-tag / CLIP-tag in buffer at ph 7.5 4/4/08 Instructions CLIP-tag Cell Surface Trial Kit, LK347 1/9

2 Materials Kit contents pcems1-clip10m-k1r Cell surface localization control plasmid: 5 µg of pcems1-clip10m-k1r dried down on a CloneSaver punch CT-488 Green fluorescent CLIP-tag substrate: 1 vial containing 20 nmol of dried CT-488 CT-547 Red fluorescent CLIP-tag substrate: 1 vial containing 20 nmol of dried CT-547 DMSO 1 vial containing 500 μl of DMSO, for dissolving CT-488 and CT-547 Instructions for use Materials required but not supplied Mammalian cells Tissue culture materials and media, transfection reagents Fluorescence microscope with suitable filter sets Related products CLIP-express plasmid: vector for transient or stable expression of CLIP-tag fusion proteins in mammalian cells CLIP-source plasmid: source of the CLIP-tag gene, codon optimized for mammalian expression, to be used as a PCR template The CT-488 substrate is also available in a quantity of 50 and 100 nmol (P/ LK341 and P/ LK342) The CT-547 substrate is also available in a quantity of 50 and 100 nmol (P/ LK343 and LK344) CLIP-cell Kits for labeling CLIP-tag fusion proteins inside live cells with blue CT-360 and CT-430, green CT-505 and CT-PF, and red CT-TMR For further details and ordering information for these products go to Storage and Handling Storage of materials supplied The dried down plasmid can be stored at room temperature for at least 3 months. After recovery from the CloneSaver material (see below), store the plasmid solution at 4 C. For long-term storage -20 C is recommended. Store the box containing CLIP-tag substrates and DMSO at - 20 C. After dissolving the CLIP-tag substrates, they should be stored at -20 C. Protect the substrates from light and moisture. With proper storage at -20 C the kit contents should be stable for at least three months. General Description of the pcems1-clip10m- K1R Plasmid The pcems1-clip10m-k1r control plasmid contains the gene encoding the eurokinin-1 receptor cloned as a fusion to the C-terminus of the CLIP-tag. The signal peptide fused to the -terminus of CLIP-tag is based on the 5HT3A serontonin receptor. The K1 receptor is a member of the G protein coupled receptor class. The CLIP-K1 receptor is inserted in the plasma membrane with the CLIP-tag exposed to the extracellular side of the membrane. When labeled with non-cell permeable CLIP-tag substrates, it gives a selective cell membrane fluorescent labeling pattern. The pcems1-clip10m-k1r plasmid was constructed by cloning the K1R gene downstream, and the signal peptide upstream of the CLIP-tag coding sequence in pcems1-clip10m, thus the receptor is fused to the C-terminus of the CLIP-tag. The pcems1-clip10m plasmid is intended for transient and stable expression of CLIP-tag gene fusions in mammalian cell lines. The plasmid contains the CMV promoter followed by the genes for the CLIP-tag and neomycin resistance separated by the IRES of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RA. After selection of stable mammalian cells for neomycin resistance, nearly all surviving colonies should therefore stably express the CLIP-tag fusion protein. An intron is also included because this is believed to improve expression levels. The plasmid contains the β-lactamase (bla, Ampicillin resistance) gene for maintenance in bacteria. A plasmid map and the sequence of the cloning region of pcems1-clip10m-k1r can be found at the end of these instructions. The complete plasmid sequence can be downloaded at Instructions for Use 1 Expression of pcems1-clip10m-k1r Read all the instructions before starting to express the CLIP-K1R fusion protein. The plasmid must be amplified in bacteria prior to use for transfections. Recovery of DA from the CloneSaver punch Sufficient DA can be recovered from the CloneSaver punch (>1 µg) to allow for transformation of bacteria Add 100 µl of sterile TE buffer (10 mm Tris-HCl, 0.1 mm EDTA, ph 8.0) to the vial with the CloneSaver punch. Make sure that the CloneSaver punch is covered by the buffer solution. Leave for 15 minutes at room temperature Mix gently by pipeting up and down and transfer the buffer from the punch into a sterile vial. This solution should be stored at 4 C The DA solution should be diluted at least 10 fold before using it for transformations as leachates from the CloneSaver material will reduce transformation effi- 2/9 Instructions CLIP-tag Cell Surface Trial Kit, LK347 4/4/08

3 ciency if it is used undiluted. The DA solution can however be used neat for PCR-reactions if required Transform the plasmid into an appropriate E. coli strain with ampicillin selection and perform a plasmid preparation using standard methods to give a plasmid stock for cloning, transfection, and long term storage. Transient expression The following protocol describes the transient expression of the control plasmid pcems1-clip10m-k1r. Genes of interest cloned into pcems1-clip10m can be used for stable or transient expression of the CLIP-tag fusion protein in the mammalian cell line of interest. For stable expression of CLIP-tag fusion proteins refer to the instructions supplied with the pcems1-clip10m plasmid (P/ 324), which can be downloaded at Expression of pcems1-clip10m-k1r can be achieved by transiently transfecting cells in culture with standard transfection protocols. The appropriate reagent and time to permit adequate expression must be empirically determined. We generally use FuGene 6 at a 3:1 DA/FuGene 6 ratio, and CLIP-tag fusion proteins can usually be observed hours post-transfection. Figure 1 shows that the CLIP-K1R fusion protein gives a selective membrane signal when labeled with non-cell permeable CLIP-vitro substrates. The pcems1-clip10m-k1r localization control plasmid has performed well in several cell lines such as CHO-K1, COS-7 and U2-OS cells. ote that the intensity of the fluorescence may vary depending on cell-line and labeling substrate used. pcems1-clip10m-k1r CLIP-K1R in CHO-K1 cells Figure 1: CLIP-K1R gives a specific membrane signal when labeled with CLIP-vitro substrate CT-547. (pcems1-clip10m-k1r in CHO-K1 cells) 2 Use of CLIP-tag substrates for live cell labeling Read all the instructions including the notes section at the end before starting to label the CLIP-K1R fusion protein. Dissolve one 20 nmol vial of CLIP-tag substrate in 20 µl of DMSO to give a labeling stock solution of 1 mm CT-488 or CT-547 in DMSO. Mix for 10 minutes until all the CLIP-tag substrate is dissolved. Store these stock solutions in the dark at +4 C, or for extended storage at -20 C. Different stock concentrations can be made, depending on your requirements. The substrates are soluble up to at least 10 mm. Stability of the labeling stock solution in DMSO is at least four weeks at +4 C or 3 months at -20 C. Labeling reaction 2.1. For labeling of mammalian cells we recommend diluting the labeling stock solution 1:200 resulting in a labeling medium containing 5 µm CT-488 or CT-547. We obtain best performance by adding the CLIP-tag substrate to complete medium, including serum. Do not prepare more medium with CLIP-tag substrate than you will consume within one hour Replace the medium on the cells expressing a CLIP-tag fusion protein with the CLIP-vitro labeling medium and incubate for 10 to 30 minutes Wash the cells three times with tissue culture medium or buffer (HBSS or PBS) incubate them in fresh medium for 5 to 10 minutes Image the cells with an appropriate filter set. CLIP-tag fusion proteins labeled with CT-488 should have an excitation maximum at 506 nm and an emission maximum at 526 nm, and can be imaged with standard fluorescein filter sets. CLIP-tag fusion proteins labeled with CT-547 should have an excitation maximum at 554 nm and an emission maximum at 568 nm, and can be imaged with standard TAMRA or Cy3 filter sets. otes Optimizing labeling The substrate concentration and incubation time can be varied depending on the experimental conditions and expression levels of the CLIP-tag fusion protein. We recommend starting with a substrate concentration between 5 and 10 μm and incubation times between 15 and 30 minutes. ote that longer incubation times may result in endocytosis of the labeled surface protein. Stability of labeling The turnover rates of the CLIP-tag fusion protein under investigation may vary widely depending on the fusion partner. We have seen half-life values ranging from less than one hour to more than 12 hours. Where protein turnover is rapid, we recommend analyzing the cells under the microscope immediately after the labeling reaction or, if the application allows it, fixing the cells directly after labeling. Fixation of cells After labeling the CLIP-tag fusion proteins, the cells can be fixed with standard fixation methods such as paraformaldehyde, ethanol, methanol, methanol/acetone etc., without loss of signal. We are not aware of any incompatibility of the CLIP-tag label with any fixation method. Counterstaining Cells can be counterstained with any live-cell dye that is compatible with the fluorescent properties of the CLIP-tag substrate for simultaneous microscopic detection. We routinely add 5 µm Hoechst to the medium that is used for the final washing step (Step 2.3) as a DA counter-stain. Counterstaining of cells is also possible after fixation and permeabilization. 4/4/08 Instructions CLIP-tag Cell Surface Trial Kit, LK347 3/9

4 Antibody labeling Antibody labeling can be performed after CLIP-tag labeling and fixation of the cells according to standard protocols without loss of the CLIP-tag signal. The fixation conditions should be selected based on experience with the protein of interest. For example some fixation methods destroy epitopes of certain proteins and therefore do not allow antibody staining afterwards. Experimental conditions that do not allow fetal calf serum If fetal calf serum has to be omitted due to the experimental setup, the staining can be done in medium without serum. Higher background levels might be observed because fetal calf serum in the labeling solution reduces the background staining. We recommend re-evaluating the dye concentration and incubation time if this is a problem. Alternatively we have found that the addition of 0.5 % BSA is helpful in some cases to block non-specific background. Troubleshooting o labeling Your protein of interest can be expressed with the CLIP-tag as either an - or a C-terminal fusion, but note that the tag needs to be exposed to the extracellular medium for labeling with CLIP-vitro substrates such as CT-488 or CT-547. If no labeling is seen, there is probably a problem with the expression of your fusion protein. Verify your transfection method to confirm that the cells contain the fusion gene of interest. If this is confirmed, check for expression of the CLIP-tag fusion protein. If no antibody against the fusion partner is available, a commercial anti-sap-tag antibody, which can be used for Western Blot detection (OpenBiosystems, anti-sap Rabbit polyclonal antibody, CAB4255) can be used to detect CLIP-tag fusion proteins. A CLIP-express plasmid may also be used as a positive control. Weak labeling may be caused by insufficient exposure of the fusion protein to the substrate. Try increasing the concentration of CLIP-tag substrate and/or the incubation time. Improving the protein expression may also improve the signal. If the protein has limited stability in the cell, it may help to analyze the samples immediately after labeling. Photobleaching is not generally a problem as the CLIP-tag substrate is very photostable. However, if you experience problems with photobleaching, addition of a commercially available anti-fade reagent may be helpful. Literature Application otes and references to publications on the use of the CLIP-tag or SAP-tag as a tool in protein labeling can be found on our website, Contact Information For technical support you can reach us directly by sending an to support@covalys.com. Covalys Biosciences AG Benkenstrasse 254 CH-4108 Witterswil Switzerland Phone: +41 (0) FAX: +41 (0) Trademarks: CLIP-tag is a trademark of Covalys Biosciences AG. CloneSaver is a trademark of The Whatman Group. FuGene 6 is a trademark of Roche Diagnostics. High background Background fluorescence may be controlled by reducing the concentration of CLIP-tag substrate used, and by shortening the incubation time. The presence of fetal calf serum or BSA during the labeling incubation should reduce non-specific binding of substrate to surfaces. In the case of transiently transfected cells, bright aggregates of non-transfected plasmid DA are sometimes seen on the cell surface. Addition of Dase I (10 µg/ml final concentration) in the labeling or washing steps may help to reduce this background. Signal strongly reduced after short time If the fluorescence signal decreases rapidly, it may be due to instability of the fusion protein. The signal may be stabilized by fixing the cells. 4/9 Instructions CLIP-tag Cell Surface Trial Kit, LK347 4/4/08

5 Plasmid Map of pcems1-clip10m-k1r All restriction enzymes shown cut at unique sites. This map can be downloaded at PvuI 6557 SspI 6992 bla rui 206 SpeI 249 dei 483 SnaBI 588 ClaI 909 EcoRV 912 P CMV BmtI 993 hei Signal peptide puc ori CLIP10m SbfI 1545 pcems1-clip10m-k1r StuI bps 2000 K1R SV40 pa PsiI 4750 HpaI 4730 XbaI 4616 SfoI 3936 ari 3936 neo IRES Flag-tag His-tag intron BlnI 3342 PmlI 3505 BamHI 2817 XhoI 2826 oti 2836 SacII /4/08 Instructions CLIP-tag Cell Surface Trial Kit, LK347 5/9

6 Cloning Region of pcems1-clip10m-k1r Unique restriction sites in the regions flanking the K1R and CLIP10m genes are displayed above the coding strand. The full DA sequence for this plasmid can be downloaded at EcoRV ClaI gagctcggat cgatatcacc atgcggctct gcatcccgca ggtgctgttg gccttgttcc >>...Signal peptide...> m r l c i p q v l l a l f BmtI hei Eco47III tttccatgct gacagggccg ggagaaggca gcgctagcat ggacaaagac tgcgaaatga >...Signal peptide...>> l s m l t g p g e g s >>...CLIP10m...> m d k d c e m 1021 agcgcaccac cctggatagc cctctgggca agctggaact gtctgggtgc gaacagggcc k r t t l d s p l g k l e l s g c e q g 1081 tgcacgagat catcttcctg ggcaaaggaa catctgccgc cgacgccgtg gaagtgcctg l h e i i f l g k g t s a a d a v e v p 1141 ccccagccgc cgtgctgggc ggaccagagc cactgatcca ggccaccgcc tggctcaacg a p a a v l g g p e p l i q a t a w l n Bsu36I cctactttca ccagcctgag gccatcgagg agttccctgt gccagccctg caccacccag a y f h q p e a i e e f p v p a l h h p 1261 tgttccagca ggagagcttt acccgccagg tgctgtggaa actgctgaaa gtggtgaagt v f q q e s f t r q v l w k l l k v v k 1321 tcggagaggt catcagcgag agccacctgg ccgccctggt gggcaatccc gccgccaccg f g e v i s e s h l a a l v g n p a a t 1381 ccgccgtgaa caccgccctg gacggaaatc ccgtgcccat tctgatcccc tgccaccggg a a v n t a l d g n p v p i l i p c h r 1441 tggtgcaggg cgacagcgac gtggggccct acctgggcgg gctcgccgtg aaagagtggc v v q g d s d v g p y l g g l a v k e w 6/9 Instructions CLIP-tag Cell Surface Trial Kit, LK347 4/4/08

7 SbfI tgctggccca cgagggccac agactgggca agcctgggct gggtcctgca ggttccacta >...CLIP10m...>> l l a h e g h r l g k p g l g >>.K1R..> g s t 1561 acacctcgga acccaatcag ttcgtgcaac cagcctggca aattgtcctt tgggcagctg n t s e p n q f v q p a w q i v l w a a BstEII cctacacggt cattgtggtg acctctgtgg tgggcaacgt ggtagtgatg tggatcatct a y t v i v v t s v v g n v v v m w i i 1681 tagcccacaa aagaatgagg acagtgacga actattttct ggtgaacctg gccttcgcgg l a h k r m r t v t n y f l v n l a f a StuI aggcctccat ggctgcattc aatacagtgg tgaacttcac ctatgctgtc cacaacgaat e a s m a a f n t v v n f t y a v h n e 1801 ggtactacgg cctgttctac tgcaagttcc acaacttctt tcccatcgcc gctgtcttcg w y y g l f y c k f h n f f p i a a v f EcoI ccagtatcta ctccatgacg gctgtggcct ttgataggta catggccatc atacatcccc a s i y s m t a v a f d r y m a i i h p 1921 tccagccccg gctgtcagcc acagccacca aagtggtcat ctgtgtcatc tgggtcctgg l q p r l s a t a t k v v i c v i w v l 1981 ctctcctgct ggccttcccc cagggctact actcaaccac agagaccatg cccagcagag a l l l a f p q g y y s t t e t m p s r 2041 tcgtgtgcat gatcgaatgg ccagagcatc cgaacaagat ttatgagaaa gtgtaccaca v v c m i e w p e h p n k i y e k v y h 2101 tctgtgtgac tgtgctgatc tacttcctcc ccctgctggt gattggctat gcatacaccg i c v t v l i y f l p l l v i g y a y t 2161 tagtgggaat cacactatgg gccagtgaga tccccgggga ctcctctgac cgctaccacg v v g i t l w a s e i p g d s s d r y h 2221 agcaagtctc tgccaagcgc aaggtggtca aaatgatgat tgtcgtggtg tgcaccttcg e q v s a k r k v v k m m i v v v c t f 4/4/08 Instructions CLIP-tag Cell Surface Trial Kit, LK347 7/9

8 2281 ccatctgcag gctgcccttc cacatcttct tcctcctgcc ctacaccaac ccagatctct a i c r l p f h i f f l l p y t n p d l 2341 acctgaagaa gtttatccag caggtctacc tggccatcat gtggctggcc atgagctcca y l k k f i q q v y l a i m w l a m s s 2401 ccatgtacaa ccccatcatc tactgctgcc tcaatgacag gttccgtctg ggcttcaagc t m y n p i i y c c l n d r f r l g f k SgrAI atgccttccg gtgctgcccc ttcatcagcg ccggcgacta tgaggggctg gaaatgaaat h a f r c c p f i s a g d y e g l e m k 2521 ccacccggta tctccagacc cagggcagtg tgtacaaagt cagccgcctg gagaccacca s t r y l q t q g s v y k v s r l e t t 2581 tctccacagt ggtgggggcc cacgaggagg agccagagga cggccccaag gccacaccct i s t v v g a h e e e p e d g p k a t p 2641 cgtccctgga cctgacctcc aactgctctt cacgaagtga ctccaagacc atgacagaga s s l d l t s n c s s r s d s k t m t e AgeI gcttcagctt ctcctccaat gtgctctccg actacaagga cgacgatgac aagaccggtg >...K1R...>> s f s f s s n v l s >>...Flag-tag...>> d y k d d d d k BamHI tgccgcgcgg cagcggcagc agccatcatc atcatcatca tcaccatcac cattaaggat >>...His-tag...>> h h h h h h h h h h XhoI oti cctgactcga gtgaggcggc cgcatagata actgatccag tgtgctggaa ttaattcgct 8/9 Instructions CLIP-tag Cell Surface Trial Kit, LK347 4/4/08

9 LIMITED USE LABEL LICESE The opening of this kit constitutes the acceptance of the following Limited Use Label License between Covalys Biosciences AG ( Covalys ) and the purchaser ( Purchaser ): 1. Research use only: The product(s) contained in this kit ( Products ) are for research use only. It is not permitted to (a) use the Products for diagnostic, prophylactic, or therapeutic purpose; (b) use the Products for manufacturing purposes; or (c) apply Products to humans or animals. 2. Implicit license to provide research services: Research on behalf of third parties ( Research Services ), for free or for consideration such as cash (for example High-Throughput Screening services), is permitted. Covalys will not claim fees on revenues generated from Research Services using the Products. 3. Transfer free of charge permitted: Transfer of the Products, derivatives or parts of thereof is permitted if the transfer is free of charge. Re-sale is forbidden. 4. Waiver of reach-through license: Covalys will not claim any reach-through licenses relating to the Purchaser s commercialization of discoveries resulting from research use of these Products or its derivatives, if (a) such discoveries by the Purchaser do not fall within the scope of the claims of patents or patent applications owned by or licensed to Covalys, and if (b) such commercialization would not be a violation of the terms of this label license. 5. Patents: The Products are partially covered by one or several of the following patents and patent applications: - PCT/EP2007/ (Labeling of Fusion Proteins with Synthetic Probes); - PCT/GB02/01636 (Methods using O6-Alkylguanine-DA-Alkyltransferases); - PCT/EP03/10889 (Substrates for O6-Alkylguanine-DA Alkyltransferase); - PCT/EP03/1085 (Protein labeling with O6-Alkylguanine-DA Alkyltransferase); - PCT/IB2004/ (Methods for protein labeling based on acyl carrier protein); - PCT/EP2005/ (Mutants of O6-Alkylguanine-DA-Alkyltransferase); - PCT/EP2005/ (Specific substrates for O6-Alkylguanine-DA-Alkyltransferase); - PCT/EP2005/ (Method for protein purification and labeling based on a chemoselective reaction). These patents and patent applications are owned by Covalys, or owned by the Ecole Polytechnique Fédérale de Lausanne (EPFL) and exclusively licensed to Covalys. 6. Implicit Agreement: By using the Products, Purchaser explicitly agrees with the terms and conditions of this Limited Use Label License. Purchasers who do not agree with the terms and conditions set forth herein can return the unopened Products at their own costs and will receive a full refund (excluding shipping, tax and handling fees). 7. Contact: For any inquiries regarding this Limited Use Label License, or for additional licenses, please contact: Covalys Biosciences AG, attn. CEO, Benkenstrasse 254, CH-4108 Witterswil, Switzerland; Tel , Fax , support@covalys.com 4/4/08 Instructions CLIP-tag Cell Surface Trial Kit, LK347 9/9