Beruflich Dokumente
Kultur Dokumente
References
7. Remove the Monoclonal ANTI-FLAG M2 solution
2 1. Brizzard, B.L., et al., Immunoaffinity purification of
and wash once with at least 0.5 ml/cm of TBS.
FLAG epitope-tagged bacterial alkaline
8. Add Anti-Mouse IgG–Peroxidase, Catalog Number phosphatase using a novel monoclonal antibody
A9044 or equivalent, to at least 0.5 ml/cm2 of TBS and peptide elution. BioTechniques, 16, 730-735
with 3% nonfat dry milk. Use the concentrations (1994)
listed in the table below. These concentrations are 2. Ciaccia, A.V., and Price, E.M., IBI FLAG Epitope, 1,
recommended starting concentrations for the 4-5 (1992)
antibodies used in the Western blot. Incubate the 3. Bjerrum, O.J., and Heegaard. N.H.H., CRC
blots with shaking at room temperature for Handbook of Immunoblotting of Proteins, Volume I,
30 minutes. Technical Descriptions, CRC Press, (1988)
p. 229-236
4. Dunbar, B.S. (ed.) Protein Blotting: A Practical
ANTI-FLAG M2 Substrate Secondary Approach, IRL Press, NY, p. 67-70 (1994)
primary antibody antibody 5. Fortin, A., et al., A 56- to 54-kilodalton non grata
(µg/ml) concentration signal in immunoblot analysis using the horseradish
0.5–10 ECL+ 1:80,000 peroxidase chemiluminescence system. Biochem.
0.5–10 ECL 1:10,000 Cell Biol., 72, 239-243 (1994)
6. Harlow, E., and Lane, D., Antibodies: A Laboratory
9. Wash the blot eight times for a total of 20 minutes Manual, Cold Spring Harbor Laboratory, Cold
in Tris Buffered Saline, with Tween 20, pH 8.0 Spring Harbor, NY, 1988, Product Code A2926
(50 mM Tris, 0.138 M NaCl, 2.7 mM KCl, pH 8.0, 7. Pampori, N.A., et al., Dilution of the chemilumin-
plus 0.05% TWEEN 20), Catalog Number T9039. escence reagents reduces the background noise
on western blots. BioTechniques, 18, 589-590
10. Develop the blots with the appropriate substrate for
(1995)
5 minutes.
11. Expose BioMax light film to the blot. Exposure FLAG and ANTI-FLAG are registered trademarks of Sigma-Aldrich
Biotechnology LP. The product designations of pFLAG, p3XFLAG,
times range from 30 seconds to 10 minutes. It is pFLAG-1, pFLAG-2, pFLAGSHIFT, pFLAG-CTS, pFLAG-ATS,
best to do a quick exposure of 10 to 30 seconds to pFLAG-MAC, pFLAG-CMV, YEpFLAG, and FLAG-BAP are
determine what exposure time is needed. If the trademarks of Sigma-Aldrich Biotechnology LP.
signal is too intense even at the short exposure
BioMax is a registered trademark of Carestream Health, Inc.
times let the signal decay from 1 to 8 hours or ECL and ECL+ are trademarks of Amersham Biosciences Ltd.
longer if necessary and then re-expose the film. Immobilon is registered trademark and Milli-Q is a trademark of the
Millipore Corp.
Immunofluorescence TWEEN is a registered trademark of Croda International PLC
Monoclonal ANTI-FLAG M2 may be used in
immunofluorescent procedures. A typical concentration
for use is 20 µg/ml.2
Product Profile
Antigenic binding site:
N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C
Troubleshooting Guide
These products and/or their use are covered by one or more of the B. Antibody License: You may only use the enclosed antibody
following patents:US 5,011,912, US 4,703,004, US 4,782,137, US for research purposes to perform a method of producing a protein in
4,851,341, EP 150126,EP 335899, JP 1983150,JP 2665359, CA which the protein is expressed in a host cell and purified by use of the
1307752. Use of these products are subject to the terms of a license antibody in accordance with a claim in one of the above patents in
provided in the product packaging, a copy of which will be provided force in a country where the use actually occurs so long as: (1) you
upon request. perform such method with a DNA expression vector licensed from
Sigma-Aldrich Co.; and (2) you do not bind (or allow others to bind)
LICENSE AGREEMENT an unlicensed antibody to any DYKDDDDK epitope of any fusion
The enclosed DNA expression vector and/or antibody are specifically protein that is produced by use of the method.
adapted for a method of producing selected protein molecules
covered by one or more of the following patents owned by Sigma- This license does not include any rights under any other patents. You
Aldrich Co.: U.S. Patent Nos. 5,011,912, 4,703,004, 4,782,137 and are not licensed to use the vector and/or antibody in any manner or
4,851,341; EP Patent No. 150,126 (Austria, Belgium, Switzerland, for any purpose not recited above. As used above, the term
France, United Kingdom, Italy, Netherlands and Sweden); EP Patent "unlicensed antibody" means any antibody which Sigma-Aldrich Co.
No. 335,899 (Belgium, Switzerland, Germany, France, United has not expressly licensed pursuant to Paragraph B, above. Sigma-
Kingdom, Italy, Luxembourg and Sweden); German Patent No. Aldrich Co. hereby expressly retains all rights in the above listed
P3584260.1; Canadian Patent No. 1,307,752; and Japanese Patent patents not expressly licensed hereunder.
Nos. 1,983,150 and 2,665,359. Your payment includes a limited
license under these patents to make only the following uses of these If the terms and conditions of this License Agreement are acceptable
products: 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
A. Vector License: You may use the enclosed vector to your acceptance to these terms and conditions.
transform cells to produce proteins containing the amino acid
sequence DYKDDDDK for research purposes provided, however, If the terms and conditions of this License Agreement are not
such research purposes do not include binding an unlicensed acceptable to you, then please return the vessel(s) unopened to
antibody to any portion of this amino acid sequence nor using such Sigma-Aldrich Co. for a complete refund of your payment.
proteins for the preparation of antibodies having an affinity for any
portion of this amino acid sequence. 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.
TD,KAA,PHC 12/08-1