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TECHNICAL BULLETIN
Product Description Preparation Instructions
The monoclonal ANTI-FLAG M2 antibody is a murine Immediately prior to use, dilute the monoclonal
derived, affinity purified IgG1 monoclonal antibody that ANTI-FLAG M2 antibody solution in Tris Buffered
binds to fusion proteins containing a FLAG peptide Saline (TBS), pH 8.0, with 3% nonfat Milk (Product
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sequence. The M2 antibody will recognize a FLAG Code T 8793). Dilutions in the described procedures
peptide sequence at the N-terminus, Met-N-terminus, are provided as guidelines. Adjust the antibody
C-terminus, or internal sites of a fusion protein. Binding concentration to maximize detection sensitivity and
of the M2 antibody is not calcium dependent. The minimize background.
monoclonal ANTI-FLAG M2 antibody is useful for
detection, identification, and capture of fusion proteins Storage
containing a FLAG peptide sequence by common Store the undiluted antibody at −20 °C in working
immunological procedures, such as Western blotting, aliquots. The product, as formulated, will not freeze
immunofluorescence, and immunoprecipitation. when stored at the recommended temperature.
The monoclonal ANTI-FLAG M2 antibody is supplied at Note: Over time, small amounts of purified antibodies
a concentration of ∼1 mg of protein per ml of solution, can precipitate due to intermolecular hydrophobic
and is formulated in 50% glycerol, 10 mM sodium interactions. If precipitate is observed in this product,
phosphate, and 150 mM NaCl, pH 7.4. The formulation briefly centrifuge the vial to pellet the precipitate.
contains no antimicrobial preservatives. Withdraw the desired volume of antibody solution from
the clear supernatant for use. This should not alter the
Antigenic binding site: performance of the purified antibody in most
N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C applications.
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3. Wash the blot in at least 0.5 ml/cm of purified 11. Expose BioMax™ Light film (Product Code
water for 2-3 minutes employing gentle agitation Z373494) to the blot for a range of times from
(50-60 rpm). several seconds up to 10 minutes. It is
recommended that a quick exposure of 10 to
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4. Block the blot with at least 0.5 ml/cm of Tris 30 seconds be performed to determine the optimal
Buffered Saline (TBS), pH 8.0, with 3% nonfat Milk exposure time needed. If the signal is too intense
(Product Code T8793) for 30 minutes at room even at the short exposure times, allow the signal
temperature, employing gentle agitation. to decay over a 1 to 8 hour period (or longer if
necessary) and then re-expose the film.
5. Remove the blocking agent and wash once with
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0.5 ml/cm of Tris Buffered Saline (TBS), pH 8.0 Indirect Immunofluorescent Cytochemical Staining
(Product Code T6664). The monoclonal ANTI-FLAG M2 antibody may be
utilized in immunocytochemical staining procedures
6. Add the desired concentration of monoclonal when used in conjunction with a labeled secondary
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ANTI-FLAG M2 antibody to the blot. A final antibody (indirect). A generic procedure for adherent
antibody concentration of 1 µg/ml (1:1,000 dilution cell staining is described, using immunofluorescence,
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of the antibody as supplied) in at least 0.5 ml/cm of employing an anti-mouse IgG-FITC conjugate as the
TBS with 3% nonfat milk is suggested. Incubate at label.
room temperature for 30 minutes employing gentle
agitation. 1. Grow and transfect cells on coverslips.
Note: Dilutions must be optimized for different 2. Fix the cells by incubation with Phosphate Buffered
substrates and systems. Saline, pH 7.4 (PBS, Product Code P3813)
containing 4% paraformaldehyde (Product Code
7. Decant off the ANTI-FLAG M2 antibody solution P6148) and 4% sucrose (Product Code S1888) for
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and wash once with at least 0.5 ml/cm of TBS, 15 minutes at room temperature.
pH 8.0.
3. Wash the fixed cells with PBS for 5 minutes.
8. Add the secondary antibody in the form of rabbit Repeat once.
anti-mouse IgG−peroxidase conjugate (Product
Code A9044) or equivalent. The concentration of 4. Permeabilize the cells by incubation with 0.25%
secondary antibody must be optimized based on TRITON X-100 (Product Code T9284) in PBS for
the substrate being used. For detection using 5 minutes.
Chemiluminescent Peroxidase Substrate-1
(Product Code CPS160), a final secondary 5. Wash the cells with PBS for 5 minutes. Repeat
antibody dilution of 1:30,000 should be employed. once.
Specifically, it is suggested that the antibody, as
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supplied, be diluted in at least 0.5 ml/cm of TBS 6. Block by incubation with 10% bovine serum
with 3% nonfat milk. Incubate the blot employing albumin (BSA, Product Code A9647) in PBS
gentle agitation at room temperature for (10% BSA/PBS) for 30 minutes at 37 °C.
30 minutes.
7. Incubate with monoclonal ANTI-FLAG M2 antibody
9. Wash the blot at least three times for a total of diluted in the range of 1:500 to 1:2,000 in
15 minutes (5 minutes per wash) in TBS with 3% BSA/PBS for 2 hours at 37 °C.
®
0.05% TWEEN 20, pH 8.0 (Product Code T9039).
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Agitate gently, employing at least 0.5 ml/cm of 8. Wash with PBS for 5 minutes. Repeat twice.
wash solution.
9. Incubate with the anti-mouse secondary antibody−
10. Develop the blot using Chemiluminescent FITC conjugate (Product Code F9137) at a 1:1,000
Peroxidase Substrate-1 (Product Code CPS160) or dilution in 3% BSA/PBS for 45 minutes at 37 °C.
an equivalent reagent for 5 minutes. Do not agitate
the blot during this incubation step. Drain briefly 10. Wash with PBS for 5 minutes. Repeat twice.
and wrap in plastic film.
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These products and/or their use are covered by one or more of the following patents: US 5,011,912; US 4,703,004; US 4,782,137; US 4,851,341;
EP 150126; EP 335899; JP 1983150; JP 2665359; CA 1307752. Use of these products is subject to the terms of a license provided in the product
packaging, a copy of which will be provided upon request. FLAG and ANTI-FLAG are registered trademarks of Sigma-Aldrich Biotechnology
LP. The product designations of pFLAG, p3XFLAG, pFLAG-1, pFLAG-2, pFLAGSHIFT, pFLAG-CTS, pFLAG-ATS, pFLAG-MAC,
pFLAG-CMV, YEpFLAG, and FLAG-BAP are trademarks of Sigma-Aldrich Biotechnology LP.
LICENSE AGREEMENT
The enclosed DNA expression vector and/or antibody are specifically adapted for a method of producing selected protein molecules covered by one or
more of the following patents owned by Sigma-Aldrich Co.: U.S. Patent Nos. 5,011,912, 4,703,004, 4,782,137 and 4,851,341; EP Patent No. 150,126 (Austria,
Belgium, Switzerland, France, United Kingdom, Italy, Netherlands and Sweden); EP Patent No. 335,899 (Belgium, Switzerland, Germany, France, United
Kingdom, Italy, Luxembourg and Sweden); German Patent No. P3584260.1; Canadian Patent No. 1,307,752; and Japanese Patent Nos. 1,983,150 and 2,665,359.
Your payment includes a limited license under these patents t o make only the following uses of these products:
A. Vector License: You may use the enclosed vector to transform cells to produce proteins containing the amino acid sequence DYKDDDDK for
research purposes provided, however, such research purposes do not include binding an unlicensed antibody to any portion of this amino acid sequence nor using
such proteins for the preparation of antibodies having an affinity for any portion of this amino acid sequence.
B. Antibody License: You may only use the enclosed antibody for research purposes to perform a method of producing a protein in which the
protein is expressed in a host cell and purified by use of the antibody in accordance with a claim in one of the above patents in force in a country where the use
actually occurs so long as: (1) you perform such method with a DNA expression vector licensed from Sigma-Aldrich Co.; and (2) you do not bind (or allow others
to bind) an unlicensed antibody to any DYKDDDDK epitope of any fusion protein that is produced by use of the method.
This license does not include any rights under any other patents. You are not licensed to use the vector and/or antibody in any manner or for any
purpose not recited above. As used above, the term "unlicensed antibody" means any antibody that Sigma-Aldrich Co. has not expressly licensed pursuant to
Paragraph B, above. Sigma-Aldrich Co. hereby expressly retains all rights in the above listed patents not expressly licensed hereunder.
If the terms and conditions of this License Agreement are acceptable to you, then you may open the vessel(s) containing the vector and/or antibody
and, through such act of opening a vessel, will have shown your acceptance to these terms and conditions.
If the terms and conditions of this License Agreement are not acceptable to you, then please return the vessel(s) unopened to Sigma-Aldrich Co. for a
complete refund of your payment.
For additional licensing information or to receive a copy of any of the above patents, please contact the Sigma-Aldrich Co. licensing department at
telephone number 314-771-5765.