SNAP-Alexa Fluor® 647 Kits
for labeling SNAP-tag™ fusion proteins in
solution or on the surface of cells
with BG-Alexa Fluor® 647; Based on
Molecular Probes’ Alexa Fluor® 647.
Product Nos. LK258 (50 nmol) and
LK259 (100 nmol)
This product contains the materials required to label a SNAP- Kit contents
tag fusion protein in solution or on the surface of living cells
• BG-Alexa Fluor® 647 - SNAP-tag substrate: 1 or 2 vials,
with the cell impermeable substrate BG-Alexa Fluor® 647.
each containing 50 nmol of dried down
This photostable fluorescent substrate is based on the Al-
BG-Alexa Fluor® 647
exa Fluor® dye Alexa Fluor® 647 and can be used with
standard 635 nm or 650 nm diode laser excitation. It has an • DMSO – One 1.5 mL vial with 500 µL of DMSO, for dis-
excitation maximum at 652 nm and an emission maximum solving the substrate
at 670 nm. The 50 nmol of BG-Alexa Fluor® 647 substrate in
each vial are sufficient to label >1.5 mL of a 20 µM SNAP-tag • Instructions for use
fusion protein solution, or to make 10 mL of a 5 µM solution
for the labeling of SNAP-tag fusion proteins on the surface of Materials required but not supplied:
cells. Separation devices for separating labeled protein in For labeling in solution:
solution from non-reacted SNAP-tag substrate, such as size
exclusion chromatography columns, are not included in • Unlabeled SNAP-tag fusion protein
• Buffer for protein dilution and labeling
The SNAP-tag is a novel tool for protein research, allowing
• Device for separation of BG labeled protein from non-
the specific, covalent attachment of virtually any molecule to
reacted SNAP-tag substrate, e.g. spin column
a protein of interest. The SNAP-tag is a polypeptide based on
mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). • UV-Vis spectrophotometer for determination of degree of
SNAP-tag substrates are derivates of benzyl purines and labeling
benzyl pyrimidines. In the labeling reaction, the substituted
For labeling on the surface of living cells:
benzyl group of the substrate is covalently attached to the
SNAP-tag. There are two steps to the use of this system: sub- • Cells expressing SNAP-fusion proteins. Your proteins of
cloning and expression of the protein of interest as a SNAP- interest can be expressed with the SNAP-tag as either an
tag fusion, and labeling of the fusion with the SNAP-tag sub- N- or a C-terminal fusion, but note that the tag needs to
strate of choice. Expression of SNAP-tag fusion proteins is be exposed to the extracellular surface of the plasma
described in the documentation supplied with SNAP-tag membrane for labeling with BG-Alexa Fluor® 647.
plasmids. The labeling of the fusion proteins with the SNAP-
tag substrate is described in this document. • Tissue culture materials and media, transfection re-
500 550 600 650 700 750 800
Figure 1. Excitation (dotted line) and emission (full line)
spectra of BG-Alexa Fluor® 647 coupled to SNAP-tag in
buffer at pH 7.5
IFU 1439 8/7/2007 Instructions SNAP-Alexa Fluor® 647 Kits; LK258,LK259 1/6
Related products Instructions for Labeling with
• Cell impermeable substrates for labeling SNAP-tag fu- BG-Alexa Fluor® 647 in Solution
sion proteins on the surface of cells with green BG-488,
BG-Alexa Fluor® 488, BG-547, BG-Alexa Fluor® 546 and Read all the instructions including the notes section at the
dark-red BG-647. end before starting to label your SNAP-tag fusion proteins.
• SNAP-source plasmids: a source of the SNAP-tag se- Dissolve the vial of SNAP-tag substrate (50 nmol) in 16.5 µL
quence, for use as a PCR template. of DMSO to give a stock solution of 3 mM SNAP-tag substrate
in DMSO. Mix for 10 minutes until all the SNAP-tag substrate
• SNAP-express plasmids: expression vectors for SNAP-tag
is dissolved. Store this stock solution in the dark at +4°C, or
for extended storage at -20°C.
• SNAP-vista kits: for the labeling of SNAP-tag fusion pro-
The dissolved SNAP-tag substrate will not precipitate when
teins for fluorescent detection on SDS-PAGE gels.
the DMSO solidifies at temperatures below 15°C.
• For further details and ordering information for these
Stability of the labeling stock solution in DMSO is at least
products go to www.covalys.com
four weeks at +4°C or 3 months at -20°C.
1 Labeling reaction
Storage and Handling See the Notes section for details of buffer composition for
SNAP-tag labeling and a calculated example.
Storage of materials supplied
1.1 Prepare a protein solution containing up to 20 µM SNAP-
Store the box containing BG-Alexa Fluor® 647 and DMSO at tag fusion protein to be labeled in an appropriate buffer
-20°C. After dissolving BG-Alexa Fluor® 647 it should be containing at least 1 mM DTT. We recommend labeling
stored at -20°C. If you use the material often it may be more at least 100 µL of protein solution per experiment.
convenient to store the dissolved BG-Alexa Fluor® 647 at
1.2 Add SNAP-tag substrate solution to a total volume of 1%
+4°C. Protect the SNAP-tag substrate from light and from
of the volume of the protein solution. Carefully pipette
moisture. With proper storage at -20°C,
the material up and down to mix, and vortex briefly.
BG-Alexa Fluor® 647 should be stable for at least three
months. 1.3 Incubate for 1 hour at 25°C in the dark. Alternatively
incubate overnight at 4°C in the dark.
Storage and handling of unlabeled SNAP-tag fusion
proteins 2 Removal of non-reacted substrate
Note: Correct storage and handling of unlabeled SNAP-tag After the labeling reaction you may wish to separate the non-
fusion proteins is essential to maintain reactivity of the reacted substrate from the labeled SNAP-tag fusion protein.
SNAP-tag prior to labeling. You can use gel filtration as well as dialysis. Please refer to
the vendor’s instructions for the separation tools you are
SNAP-tag fusion proteins can be purified before labeling, but
the labeling reaction also works in non-purified protein solu-
tions including cell lysates.
Add 1 mM DTT to buffers used for the storage of unlabeled
SNAP-tag fusion proteins. Unlabeled protein samples should We recommend the routine addition of 1 mM DTT to all buff-
be stored at -20°C, or at -80°C for long-term storage. It may ers for used for handling, labeling and storage of the SNAP-
be best to shock freeze by rapidly cooling the sample to a tag. The stability of the SNAP-tag is improved in the presence
temperature significantly below 0°C. Handling at tempera- of reducing agents; however it can also be labeled in their
tures above 0°C should be minimized by thawing the unla- absence.
beled protein samples shortly before use, and keeping them The SNAP-tag labeling reaction works well between pH 5.0
on ice until just before the labeling reaction. and pH 10.0
If a particular fusion protein requires buffers without reduc- The salt concentration may need to be optimized for your
ing agents, pay particular attention to minimize all handling particular fusion protein, although the SNAP-tag labeling
steps of the protein above 4°C before the labeling reaction. reaction has been shown to work at a broad range of ionic
The SNAP-tag itself is tolerant of a wide range of buffers. The strengths (NaCl concentrations between 15 mM and 1 M). If
requirements of your fusion partner should dictate the selec- the buffer conditions used for labeling lead to insolubility of
tion of the buffer. From our experience, the following storage your protein, we recommend testing a range of pH and ionic
buffer composition gives good performance, especially when strengths. For a range of fusion proteins we have found ionic
freezing protein material: pH between 7.0 and 8.0, monova- strengths for monovalent salts (e.g. sodium chloride) from
lent salts (e.g. sodium chloride) between 50 mM and 50 mM to 250 mM helpful.
250 mM, at least 1 mM DTT. Non-ionic detergents can be Where stickiness of the fusion protein is a problem we rec-
added if required, but ionic detergents should be avoided ommend adding Tween 20 at a final concentration of 0.05%
because they reduce the activity of the SNAP-tag. Many pro- to 0.1%. At this concentration Tween 20 does not affect the
teins benefit from the addition of glycerol for frozen storage, performance of the SNAP-tag.
typically 20% v/v.
2/6 Instructions SNAP-Alexa Fluor® 647Kits; LK258,LK259 IFU1438, 8/7/2007
The molar concentration of protein (c) can be calculated as where 0.13 is the absorbance of BG-Alexa Fluor® 647
follows: substrate at 280 nm as a proportion of its absorbance
at 650 nm.
conc (mg / mL)
c = 4. Calculate the degree of labeling:
Mw ( g / mol )
For example, a 37 kD protein at 0.5 mg/mL has a molar Label conc.
Degree of labeling =
concentration of 13.5 µM as calculated below: Protein conc.
0.5 (mg /mL) The degree of labeling should be between 0.5 and 1.0. Ow-
c= = 13.5 × 10 − 6 M
37000 ( g / mol ) ing to the intrinsic imprecision of protein assays (by absorb-
ance at 280 nm or by colorimetric or fluorimetric methods),
If labeling is not sufficient after incubation at the times rec- values up to 1.3 are acceptable. If the degree of labeling is
ommended, try adjusting time and volumes as described in significantly below 0.5, retry the labeling or refer to “Under-
the Troubleshooting section under the heading ‘Labeling’. labeling” in the Troubleshooting section for further sugges-
tions. If the degree of labeling is significantly above 1.0, the
separation of labeled protein and non-reacted substrate may
not have worked. Refer to “Inefficient removal of non-reacted
Determination of the Degree of Labeling substrate” in the Troubleshooting section for suggested
Note: the maximum degree of labeling expected with the
SNAP-tag is one molecule of label per molecule of fusion
Storage and Handling of Labeled SNAP-tag
Measurement of the degree of labeling Fusion Proteins
If your SNAP-tag fusion protein was purified (i.e. is not con-
taminated with other proteins), the non-reacted substrate Storage of the labeled protein
has been removed, and it is at a concentration of 2 µM pro- Labeled SNAP-tag fusion proteins should be stored at condi-
tein or above, it is possible to use UV-Vis spectroscopy to tions that are compatible with the fusion partner. Typical
determine the degree of labeling (ratio of SNAP-tag la- storage conditions are concentration of 1 mg/mL or less
bel / SNAP-tag fusion protein) according to the following protein at 4°C protected from light. Sodium azide may be
steps: added to 2 mM final concentration. Under these conditions
1. Take a sample of the labeled SNAP-tag fusion protein in the labeled protein should be stable at 2–6°C for several
a suitable buffer at pH 7.0 to 8.0 and measure the ab- months, depending on the fusion partner. If freezing is re-
sorbance in a microcuvette with 1 cm pathlength at quired, it may be best to shock freeze by rapidly cooling the
650 nm (A650), and at 280 nm (A280). If either value sample to a temperature significantly below 0°C.
measured is above 1.5 absorbance units, you should Note: AVOID REPEATED FREEZING AND THAWING. PROTECT
further dilute your sample to get an absorbance in the FROM LIGHT.
range from 0.1 to 1.5.
2. Calculate the molar concentration of the label: Handling of the labeled protein
A 650 × dilution factor SNAP-tag fusion proteins labeled with BG-Alexa Fluor® 647
Label conc. = should have a fluorescence excitation maximum at 652 nm
239'000 and an emission maximum at 670 nm, and can be used with
where 239’000 M-1 cm-1 is the approximate molar ex- standard 635 nm or 650 nm diode laser excitation.
tinction coefficient of the BG--Alexa Fluor® 647 dye at
The SNAP-tag label is linked to the protein by a covalent
bond. Therefore the conjugates are highly stable. DTT or
3. Determine the molar concentration of protein in the other reducing agent is not required in the storage buffers.
sample: You can do this by an appropriate protein assay As the fusion protein is covalently labeled, labeled protein
or by absorbance at 280 nm. The molar extinction coef- may be detected after harsh or even denaturing treatments,
ficient of the SNAP-tag sequence is ≈ 21300 M-1 cm-1. such as chromatographic or electrophoretic separation in-
The molar extinction coefficient for proteins at 280 nm cluding SDS-PAGE.
varies, so we recommended that you determine the ex-
tinction coefficient for your protein based on a sample of
your protein of known concentration. Determine the
Combined extinction coefficient for the SNAP-tag fusion Troubleshooting for Labeling in Solution
protein by adding both values. From this Combined ex-
tinction coefficient you can calculate the protein con- Labeling reaction
centration as follows:
If solubility problems occur with your SNAP-tag fusion protein,
( A − ( A650 × 0.13)) × dilutionfactor
Protein conc. = 280
we recommend testing a range of pH and ionic strengths.
Combined extinction coefficient The salt concentration may also need to be optimized for
your particular fusion protein.
IFU 1439 8/7/2007 Instructions SNAP-Alexa Fluor® 647 Kits; LK258,LK259 3/6
Where stickiness of the fusion protein is a problem we rec- Dissolve the vial of SNAP-tag substrate (50 nmol) in 50 µL of
ommend adding Tween 20 at a final concentration of 0.05% DMSO to give a stock solution of 1 mM SNAP-tag substrate in
to 0.1%. The SNAP-tag activity is not affected by this concen- DMSO. Mix for 10 minutes until all the SNAP-tag substrate is
tration of Tween 20. dissolved. Store this stock solution in the dark at +4°C, or for
extended storage at -20°C.
If exhaustive labeling of a protein sample is not achieved
using the recommended conditions, try the following protocol Stability of the labeling stock solution in DMSO is at least
modifications: Double the incubation time to two hours total four weeks at +4°C or 3 months at -20°C.
at 25°C or to 24 hours at 4°C; or halve the volume of pro-
1 Labeling reaction
tein solution labeled (50 µL of a solution containing up to
20 µM SNAP-tag fusion protein). Both approaches may be See the Notes section for details of buffer composition for
combined. If you still have poor labeling results, we recom- SNAP-tag labeling and a calculated example.
mend checking the activity of the SNAP-tag (see Under-
labeling). 1.1 Dilute the labeling Stock Solution 1:200 in tissue culture
medium resulting in a labeling solution of 5 µM. We ob-
If your fusion protein is particularly sensitive to degradation tain best performance by adding the SNAP-vitro sub-
or to loss of activity, you can try reducing the labeling time or strate to complete medium, including serum. Do not
decreasing the labeling temperature. If you label at 4°C we prepare more medium with SNAP-tag substrate than you
recommend over night incubation. will consume within one hour.
1.2 Replace the medium on the cells expressing a SNAP-tag
fusion protein with the SNAP-vitro labeling medium and
If the degree of labeling is significantly below 0.5 the labeling incubate for 10 minutes.
reaction was probably inefficient. A number of reasons can
1.3 Wash the cells 3 times with tissue culture medium.
lead to such inefficient labeling.
1.4 Image the cells with an appropriate filter set. SNAP-tag
If you added the substrate at less than 1% of the volume of
fusion proteins labeled with BG-Alexa Fluor® 647
protein solution, this will slow down the reaction rate. At
should have an excitation maximum at 652 nm and an
lower substrate concentrations the labeling time should be
emission maximum at 670 nm, and can be imaged with
increased to 2 hours.
standard 635 nm or 650 nm diode laser excitation.
The activity of the SNAP-tag may have been partially or com-
We recommend routinely labeling one well of non-transfected
pletely lost. This may be due to extended storage of non-
or mock-transfected cells for comparison.
reacted SNAP-tag fusion proteins at 4°C or above. The sensi-
tivity of the SNAP-tag to inactivation is also significantly in-
creased if no reducing agent is added (such as DTT at 2 Notes
1 mM). Optimizing labeling
If you believe that the activity of the SNAP-tag is affected, we The substrate concentration and incubation time can be
recommend analyzing a small fraction of it on a PAGE gel varied depending on the experimental conditions and ex-
using one of Covalys’ SNAP-vista kits to confirm that the pression levels of the SNAP-tag fusion protein. Best results
SNAP-tag is active. are usually obtained at concentrations between 5 and 10 µM
If you encounter problems with the activity we recommend and incubation times between 5 and 15 minutes. Note that
thawing another sample of your protein or re-expressing and longer incubation times may result in some labeling inside
re-purifying the SNAP-tag fusion protein following the advice the cells due to slight cell permeability of the substrates.
given in the SNAP-tag plasmid instructions. Stability of labeling
Inefficient removal of non-reacted substrate The turnover rates of the SNAP-tag fusion protein under
investigation may vary widely depending on the fusion part-
Although we have had good success in removing non-reacted ner. We have seen half-life values ranging from less than one
substrate from labeled SNAP-tag fusion proteins using gel hour to more than 12 hours. Where protein turnover is rapid,
filtration and spin columns, it is possible that trace amounts we recommend analyzing the cells under the microscope
may remain in the protein solution after your chosen separa- immediately after the labeling reaction or, if the application
tion step. The presence of non-reacted substrate will result in allows it, fixing the cells directly after labeling.
erroneously high calculated values for the degree of labeling
(see Determination of the Degree of Labeling). Remaining Fixation of cells
traces of non-reacted substrate can be removed by repeating We have shown that after labeling the SNAP-tag fusion pro-
the chosen separation step or by extensive dialysis. teins, cells can be fixed with para-formaldehyde without loss
of signal. We are not aware of any incompatibility of the
SNAP-tag labels with other fixation methods.
Instructions for Labeling with Counterstaining
BG-Alexa Fluor® 647 on the surface of living Cells can be counterstained with any live-cell dye that is
cells compatible with the fluorescent properties of the SNAP-tag
substrate for simultaneous microscopic detection. We rou-
Read all the instructions including the notes section at the tinely add 5 µM Hoechst 33342 to the medium that is used
end before starting to label your SNAP-tag fusion proteins. for the first wash (Step 1.3) as a DNA counter-stain and leave
this on the cells for 2 minutes, prior to completing the wash
4/6 Instructions SNAP-Alexa Fluor® 647Kits; LK258,LK259 IFU1438, 8/7/2007
steps. Counterstaining of cells is also possible after fixation Signal strongly reduced after short time
If the fluorescence signal decreases rapidly, it may be due to
Antibody labeling instability of the fusion protein. The signal may be stabilized
by fixing the cells.
Antibody labeling after SNAP-tag labeling and fixation of the
cells should be possible according to standard protocols Photobleaching is not generally a problem as the SNAP-tag
without loss of the SNAP-tag signal. The fixation conditions substrate is very photostable. However, if you experience
should be selected based on experience with the protein of problems with photobleaching, addition of a commercially
interest. For example some fixation methods destroy epi- available anti-fade reagent may be helpful.
topes of certain proteins and therefore do not allow antibody
Experimental conditions that do not allow fetal calf Literature
If fetal calf serum has to be omitted due to the experimental Application Notes and references to publications on the use
setup, the labeling can be done in medium without serum. of the SNAP-tag or AGT as a tool in protein labeling can be
Higher background levels might be observed because fetal found on our website, www.covalys.com.
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. The addition of
0.5% BSA may be helpful in some cases to block non-specific Contact Information
For technical support you can reach us directly by sending an
email to firstname.lastname@example.org.
Troubleshooting for Labeling of Cells Covalys Biosciences AG
Your protein of interest can be expressed with the SNAP-tag Phone: +41 (0)61 725 20 50
as either an N- or a C-terminal fusion, but note that the tag FAX: +41 (0)61 725 20 55
needs to be exposed to the extracellular medium for labeling
with BG-Alexa Fluor® 647.
If no labeling is seen, there is probably a problem with the
expression of your fusion protein. Verify expression of the Trademarks: SNAP-tag is a trademark of Covalys Biosciences
SNAP-tag fusion protein using SNAP-vista for detection of AG. Cy3 is a trademark of Amersham Biosciences.
SNAP-tag fusion proteins directly on an SDS-PAGE gel or by
western blotting. If no antibody against the fusion partner is Alexa Fluor is a registered Trademark of Invitrogen Corpora-
available, a commercial anti-AGT antibody can be used tion.
(Chemicon, mouse Anti-MGMT (O6-methylguanine-DNA me-
thyltransferase) Monoclonal Antibody, Clone MT3.1 Catalog
Weak labeling may be caused by insufficient exposure of the
fusion protein to the substrate. Try increasing the concentra-
tion of SNAP-tag substrate and/or the incubation 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.
Background fluorescence may be controlled by reducing the
concentration of SNAP-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.
IFU 1439 8/7/2007 Instructions SNAP-Alexa Fluor® 647 Kits; LK258,LK259 5/6
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 Molecular Probes, Inc. (“Molecular Probes”) on the one side and the purchaser (“Purchaser”) on the other side.
By using the Products, Purchaser explicitly agrees with the terms and conditions of the 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).
This product is provided under an agreement between Molecular Probes, Inc. and Covalys Biosciences AG, and the manufac-
ture, use, sale or import of this product is subject to one or more US patents and corresponding non-US equivalents, owned by
Molecular Probes, Inc. (a wholly-owned subsidiary of 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 only in research for
directed covalent labeling of proteins conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer
must not use the product or its components (1) in manufacturing; (2) to provide a service, information, or data; (3) for thera-
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than research, contact Molecular Probes, Inc., Business Development, 29851 Willow Creek Road, Eugene, OR 97402, Tel:
(541) 465-8300. Fax: (541) 335-0354.
This product further is covered by one or several of the following patents and patent applications:
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.
Contact: For any inquiries regarding this Limited Use Label License which are not related to the Molecular Probes labels en-
closed, please contact:
Covalys Biosciences AG, attn. CEO, Benkenstrasse 254, CH-4108 Witterswil, Switzerland;
Tel. +41 61 725 20 50, Fax +41 61 725 20 55, email: email@example.com
6/6 Instructions SNAP-Alexa Fluor® 647Kits; LK258,LK259 IFU1438, 8/7/2007