CLIP-tag Cell Surface Trial Kit

(1)

CLIP-tag Cell Surface Trial Kit

for evaluating specific cell-surface CLIP-tag

labeling with novel cell lines and imaging

technologies

Product No. LK347

Introduction

This kit enables users to rapidly evaluate selective cell-surface labeling using the CLIP-tag technology with their cell lines and microscopy equipment. This kit contains the re-agents required to selectively label cell-surface exposed CLIP-tag fusion proteins in living cells with non-cell perme-able 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 plas-mid, pCEMS1-CLIP10m-NK1R, for the transient or stable expression of the tagged Neurokinin-1 receptor (NK1R) in mammalian cells. The CLIP–NK1 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-DNA-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 SNAP-tag, the benzylcytosine substrates form a separate class of substrates, different from the benzylguanine substrates recognized by SNAP-tag. CLIP-tag and SNAP-tag can be used for orthogonal simulta-neous 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 de-scribed 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 prop-erty of Atto-Tec, Siegen, Germany (www.atto-tec.de). For further information please contact Atto-Tec directly.

0 20 40 60 80 100 400 450 500 550 600 650 Wavelength [nm]

Figure 1. Excitation (dotted line) and emission spectra of label 488 coupled to SNAP-tag / CLIP-tag in buffer at pH 7.5

N O3S N NaO3S N N O H N NH2 O Figure 2. Structure of CT-547 (MW 850.98) 0 20 40 60 80 100 440 490 540 590 640 690 Wavelength [nm]

Figure 3. Excitation (dotted line) and emission spectra of label 547 coupled to SNAP-tag / CLIP-tag in buffer at pH 7.5

(2)

Materials

Kit contents

• pCEMS1-CLIP10m-NK1R– Cell surface localization con-trol plasmid: 5 µg of pCEMS1-CLIP10m-NK1R 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 con-taining 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 re-agents

• Fluorescence microscope with suitable filter sets

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 CLIPtag 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-NK1R Plasmid

The pCEMS1-CLIP10m-NK1R control plasmid contains the gene encoding the Neurokinin-1 receptor cloned as a fusion to the C-terminus of the CLIP-tag. The signal peptide fused to the N-terminus of CLIP-tag is based on the 5HT3A serontonin receptor. The NK1 receptor is a member of the G protein coupled receptor class.

The CLIP-NK1 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-NK1R plasmid was constructed by cloning the NK1R gene downstream, and the signal peptide upstream of the CLIP-tag coding sequence in pCEMS1-CLIP10m, thus the receptor is fused to the C-termi-nus of the CLIP-tag. The pCEMS1-CLIP10m plasmid is in-tended 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 encepha-lomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA. 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 be-cause 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-NK1R can be found at the end of these instructions. The complete plasmid sequence can be downloaded at www.covalys.com.

Instructions for Use

1

Expression of pCEMS1-CLIP10m-NK1R

Read all the instructions before starting to express the CLIP-NK1R fusion protein. The plasmid must be amplified in bacteria prior to use for transfections.

Recovery of DNA from the CloneSaver

™

punch

Sufficient DNA can be recovered from the CloneSaver™ punch (>1 µg) to allow for transformation of bacteria. 1.1.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 tem-perature.

1.2.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.

1.3.The DNA solution should be diluted at least 10 fold before using it for transformations as leachates from the CloneSaver™ material will reduce transformation

(3)

effi-ciency if it is used undiluted. The DNA solution can how-ever be used neat for PCR-reactions if required.

1.4.Transform the plasmid into an appropriate E. coli strain with ampicillin selection and perform a plasmid prepara-tion 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-NK1R. Genes of inter-est 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/N 324), which can be downloaded at www.covalys.com

Expression of pCEMS1-CLIP10m-NK1R can be achieved by transiently transfecting cells in culture with standard trans-fection protocols. The appropriate reagent and time to permit adequate expression must be empirically determined. We generally use FuGene 6™ at a 3:1 DNA/FuGene 6 ratio, and CLIP-tag fusion proteins can usually be observed 16-24 hours post-transfection. Figure 1 shows that the CLIP-NK1R fusion protein gives a selective membrane signal when la-beled with non-cell permeable CLIP-vitro substrates. The pCEMS1-CLIP10m-NK1R localization control plasmid has performed well in several cell lines such as CHO-K1, COS-7 and U2-OS cells. Note that the intensity of the fluorescence may vary depending on cell-line and labeling substrate used.

Figure 1: CLIP-NK1R gives a specific membrane signal when labeled with CLIP-vitro substrate CT-547.

(pCEMS1-CLIP10m-NK1R 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-NK1R 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 re-quirements. 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 con-sume within one hour.

2.2.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.

2.3.Wash the cells three times with tissue culture medium or buffer (HBSS or PBS) incubate them in fresh medium for 5 to 10 minutes.

2.4.Image the cells with an appropriate filter set. CLIP-tag fusion proteins labeled with CT-488 should have an ex-citation maximum at 506 nm and an emission maxi-mum at 526 nm, and can be imaged with standard fluo-rescein 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 im-aged with standard TAMRA or Cy3 filter sets.

Notes

Optimizing labeling

The substrate concentration and incubation time can be varied depending on the experimental conditions and ex-pression levels of the CLIP-tag fusion protein. We recom-mend starting with a substrate concentration between 5 and 10 μM and incubation times between 15 and 30 minutes. Note 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 inves-tigation 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 imme-diately 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 para-formaldehyde, ethanol, methanol, methanol/acetone etc., without loss of signal. We are not aware of any incompatibil-ity 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 rou-tinely add 5 µM Hoechst 33342 to the medium that is used for the final washing step (Step 2.3) as a DNA counter-stain. Counterstaining of cells is also possible after fixation and permeabilization.

pCEMS1-CLIP10m-NK1R CLIP-NK1R in CHO-K1 cells

(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 epi-topes 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

No labeling

Your protein of interest can be expressed with the CLIP-tag as either an N- 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-SNAP-tag antibody, which can be used for Western Blot detection

(OpenBiosys-tems, anti-SNAP 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 concentra-tion of CLIP-tag substrate and/or the incubaconcentra-tion time. Im-proving 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.

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 plas-mid DNA are sometimes seen on the cell surface. Addition of DNase 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.

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 Notes and references to publications on the use of the CLIP-tag or SNAP-tag as a tool in protein labeling can be found on our website, www.covalys.com.

Contact Information

For technical support you can reach us directly by sending an email to [email protected]. Covalys Biosciences AG Benkenstrasse 254 CH-4108 Witterswil Switzerland Phone: +41 (0)61 725 20 50 FAX: +41 (0)61 725 20 55

www.covalys.com

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.

(5)

Plasmid Map of pCEMS1-CLIP10m-NK1R

All restriction enzymes shown cut at unique sites. This map can be downloaded at www.covalys.com.

pCEMS1-CLIP10m-NK1R

7113 bps 1000 2000 3000 4000 5000 6000 700 NruI 206 SpeI 249 NdeI 483 SnaBI 588 ClaI 909 EcoRV 912 BmtI 993 NheI 993 SbfI 1545 StuI 1741 BamHI 2817 XhoI 2826 NotI 2836 SacII 2991 BlnI 3342 PmlI 3505 NarI 3936 SfoI 3936 XbaI 4616 HpaI 4730 PsiI 4750 PvuI 6557 SspI 6992 P CMV Signal peptide CLIP10m NK1R Flag-tag His-tag intron IRES neo SV40 pA pUC ori bla

(6)

Cloning Region of pCEMS1-CLIP10m-NK1R

Unique restriction sites in the regions flanking the NK1R and CLIP10m genes are displayed above the coding strand. The full DNA sequence for this plasmid can be downloaded at www.covalys.com.

EcoRV

ClaI

901 gagctcggat cgatatcacc atgcggctct gcatcccgca ggtgctgttg gccttgttcc

>>...Signal peptide...>

m r l c i p q v l l a l f

BmtI

---+

NheI

Eco47III

961 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

>...CLIP10m...>

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

>...CLIP10m...>

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

>...CLIP10m...>

a p a a v l g g p e p l i q a t a w l n

Bsu36I

1201 cctactttca ccagcctgag gccatcgagg agttccctgt gccagccctg caccacccag

>...CLIP10m...>

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

>...CLIP10m...>

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

>...CLIP10m...>

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

>...CLIP10m...>

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

>...CLIP10m...>

(7)

SbfI

1501 tgctggccca cgagggccac agactgggca agcctgggct gggtcctgca ggttccacta

>...CLIP10m...>>

l l a h e g h r l g k p g l g

>>.NK1R..>

g s t

1561 acacctcgga acccaatcag ttcgtgcaac cagcctggca aattgtcctt tgggcagctg

>...NK1R...>

n t s e p n q f v q p a w q i v l w a a

BstEII

1621 cctacacggt cattgtggtg acctctgtgg tgggcaacgt ggtagtgatg tggatcatct

>...NK1R...>

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

>...NK1R...>

l a h k r m r t v t n y f l v n l a f a

StuI

1741 aggcctccat ggctgcattc aatacagtgg tgaacttcac ctatgctgtc cacaacgaat

>...NK1R...>

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

>...NK1R...>

w y y g l f y c k f h n f f p i a a v f

EcoNI

1861 ccagtatcta ctccatgacg gctgtggcct ttgataggta catggccatc atacatcccc

>...NK1R...>

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

>...NK1R...>

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

>...NK1R...>

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

>...NK1R...>

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

>...NK1R...>

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

>...NK1R...>

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

>...NK1R...>

(8)

2281 ccatctgcag gctgcccttc cacatcttct tcctcctgcc ctacaccaac ccagatctct

>...NK1R...>

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

>...NK1R...>

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

>...NK1R...>

t m y n p i i y c c l n d r f r l g f k

SgrAI

2461 atgccttccg gtgctgcccc ttcatcagcg ccggcgacta tgaggggctg gaaatgaaat

>...NK1R...>

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

>...NK1R...>

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

>...NK1R...>

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

>...NK1R...>

s s l d l t s n c s s r s d s k t m t e

AgeI

2701 gcttcagctt ctcctccaat gtgctctccg actacaagga cgacgatgac aagaccggtg

>...NK1R...>>

s f s f s s n v l s

>>...Flag-tag...>>

d y k d d d d k

BamHI

2761 tgccgcgcgg cagcggcagc agccatcatc atcatcatca tcaccatcac cattaaggat

>>...His-tag...>>

h h h h h h h h h h

XhoI

NotI

-+---

(9)

LIMITED USE LABEL LICENSE

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 commerciali-zation 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/EP2007/057597 (Labeling of Fusion Proteins with Synthetic Probes); - PCT/GB02/01636 (Methods using O6-Alkylguanine-DNA-Alkyltransferases);

- PCT/EP03/10889 (Substrates for O6-Alkylguanine-DNA Alkyltransferase); - PCT/EP03/1085 (Protein labeling with O6-Alkylguanine-DNA Alkyltransferase); - PCT/IB2004/001733 (Methods for protein labeling based on acyl carrier protein);

- PCT/EP2005/050899 (Mutants of O6-Alkylguanine-DNA-Alkyltransferase);

- PCT/EP2005/050900 (Specific substrates for O6-Alkylguanine-DNA-Alkyltransferase);

- PCT/EP2005/054114 (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 Prod-ucts 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;