Monoclonal ANTIFLAG® M2 Clone M2
produced in mouse affinity isolated antibody
Catalog Number F1804
Storage temperature –20 °C
TECHNICAL BULLETIN
Product Description
Monoclonal ANTIFLAG M2 is a mouse derived affinity
purified IgG1 monoclonal antibody that binds to fusion
proteins containing a FLAGÒ peptide sequence1 The
M2 antibody will recognize a FLAG peptide sequence
at the Nterminus MetNterminus Cterminus or
internal sites of a fusion protein Binding of the M2
antibody is not calcium dependent
Monoclonal ANTIFLAG M2 is useful for detection
identification and capture of fusion proteins containing
a FLAG peptide sequence by common immunological
procedures such as Western blotting
immunofluorescence and immunoprecipitation
Reagent
Supplied at a concentration of ~1 mg of protein per mL
of solution and formulated in 50 glycerol 10 mM
sodium phosphate and 150 mM NaCl pH 74 The
formulation contains no antimicrobial preservatives
Antigenic binding site
NAspTyrLysAspAspAspAspLysC
Specificity The monoclonal antibody detects only the
target protein band(s) on a Western blot from an E coli
plant or mammalian crude cell lysate
Sensitivity The monoclonal antibody detects as little as
2 ng of target protein by dot blot The Western blot is
tested down to 10 ng but may detect lower using the
procedure detailed below
Precautions and Disclaimer
This product is for R&D use only not for drug
household or other uses Please consult the Material
Safety Data Sheet for information regarding hazards
and safe handling practices
Preparation Instructions
Immediately prior to use dilute the monoclonal
antibody in Tris buffered saline (TBS) pH 80 with 3
nonfat Milk Catalog Number T8793 Dilutions in the
described procedures are provided as guidelines
Adjust the antibody concentration to maximize
detection sensitivity and minimize background
Storage
Store the undiluted antibody at –20 °C in working
aliquots The product as formulated will not freeze
when stored at the recommended temperature
Note Over time small amounts of purified antibodies
can precipitate due to intermolecular hydrophobic
interactions If precipitate is observed in this product
briefly centrifuge the vial to pellet the precipitate
Withdraw the desired volume of antibody solution from
the clear supernatant for use This should not alter the
performance of the purified antibody in most
applications
Procedures
Western Blot Immunostaining25
Note this procedure is based on chemiluminescent
detection using Chemiluminescent Peroxidase
Substrate1 Catalog Number CPS160 Dilutions of
both primary and secondary antibodies may require
optimization when using other substrates or conditions
1 Separate fusion proteins containing a FLAG
peptide sequence from sample lysates using a
standard SDSPAGE protocol Load 25–10 mg of
total protein lysate per lane
2 Transfer proteins from the gel to a nitrocellulose
membrane ImmobilonÒP or other polyvinylidene
difluoride (PVDF) membrane The PVDF
membrane may provide greater downstream
sensitivity
2
3 Wash the blot in at least 05 mLcm2 of purified
water for 2–3 minutes employing gentle agitation
(50–60 rpm)
4 Block the blot with at least 05 mLcm2 of Tris
Buffered Saline (TBS) pH 80 with 3 nonfat milk
for 30 minutes at room temperature employing
gentle agitation
5 Remove the blocking agent and wash once with
05 mLcm2 of Tris buffered saline pH 80 Catalog
Number T6664
6 Add the desired concentration of monoclonal
antibody to the blot A final antibody concentration
of 1 mgmL (11000 dilution of the antibody as
supplied) in at least 05 mlcm2 of TBS with 3
nonfat milk is suggested Incubate at room
temperature for 30 minutes employing gentle
agitation
Note Dilutions must be optimized for different
substrates and systems
7 Decant off the Monoclonal ANTIFLAG M2 solution
and wash once with at least 05 mlcm2 of TBS
pH 80
8 Add the secondary antibody in the form of Anti
Mouse IgGPeroxidase Catalog Number A9044 or
equivalent The concentration of secondary
antibody must be optimized based on the substrate
being used For detection using Chemiluminescent
Peroxidase Substrate1 a final secondary antibody
dilution of 130000 should be employed
Specifically it is suggested the antibody as
supplied be diluted in at least 05 mLcm2 of TBS
with 3 nonfat milk Incubate the blot employing
gentle agitation at room temperature for
30 minutes
9 Wash the blot at least three times for a total of
15 minutes (5 minutes per wash) in TBS with
005 TWEEN® 20 pH 80 Catalog Number
T9039 Agitate gently employing at least
05 mlcm2 of wash solution
10 Develop the blot using Chemiluminescent
Peroxidase Substrate1 or an equivalent reagent
for 5 minutes Do not agitate the blot during this
incubation step Drain briefly and wrap in plastic
film
11 Expose BioMax® Light film Catalog Number
Z373494 to the blot for a range of times from
several seconds up to 10 minutes It is
recommended that a quick exposure of
10–30 seconds be performed to determine the
optimal exposure time needed If the signal is too
intense even at the short exposure times allow the
signal to decay over a 1–8 hour period (or longer if
necessary) and then reexpose the film
Indirect Immunofluorescent Cytochemical Staining
Monoclonal ANTIFLAG M2 may be utilized in
immunocytochemical staining procedures when used in
conjunction with a labeled secondary antibody
(indirect)6 A generic procedure for adherent cell
staining is described using immunofluorescence
employing an AntiMouse IgGFITC conjugate as the
label
1 Grow and transfect cells on coverslips
2 Fix the cells by incubation with phosphate buffered
saline pH 74 Catalog Number P3813 containing
4 paraformaldehyde Catalog Number P6148
and 4 sucrose Catalog Number S1888 for
15 minutes at room temperature
3 Wash the fixed cells with PBS for 5 minutes
Repeat once
4 Permeabilize the cells by incubation with 025
TRITON™ X100 Catalog Number T9284 in PBS
for 5 minutes
5 Wash the cells with PBS for 5 minutes Repeat
once
6 Block by incubation with 10 bovine serum
albumin Catalog Number A9647 in PBS
(10 BSAPBS) for 30 minutes at 37 °C
7 Incubate with Monoclonal ANTIFLAG M2 diluted in
the range of 1500 to 12000 in 3 BSAPBS for
2 hours at 37 °C
8 Wash with PBS for 5 minutes Repeat twice
9 Incubate with the secondary antibody AntiMouse
IgG FITC Catalog Number F9137 at a 11000
dilution in 3 BSAPBS for 45 minutes at 37 °C
10 Wash with PBS for 5 minutes Repeat twice
3
11 Mount coverslips with cells side down on glass
slides using a small drop of mounting medium such
as polyvinyl alcohol for semipermanent mounting
The inclusion of an antifading agent like DABCOÒ
in the mounting medium (25–100 mgml for
example Catalog Number 10981) is strongly
recommended Seal coverslips to glass slides (eg
with nail polish)
12 Examine by fluorescence microscopy FITC has an
absorption maximum at 492 nm with an emission
maximum at 520 nm
Immunoprecipitation (IP)
Monoclonal ANTIFLAG M2 may be used in IP
procedures when used in conjunction with an insoluble
carrier matrix such as a Protein G resin Alternatively
EZview™ Red Protein G Affinity Gel Catalog Number
E3403 or the Protein G Immunoprecipitation Kit
Catalog Number IP50 may be used
EZview Red ANTIFLAG M2 Affinity Gel Catalog
Number F2426 or ANTIFLAG M2 Affinity Gel Catalog
Number A2220 may be utilized directly for IP See
reference 5 for general protocols
Enzyme Immunoassay (EIA)
Monoclonal ANTIFLAG M2 may be used in EIA
procedures Typically a fusion protein containing a
FLAG peptide sequence is directly adsorbed (or
otherwise presented) within the wells of a multiwell
polystyrene plate The Monoclonal ANTIFLAG M2
antibody may be diluted up to 150000 for subsequent
incubation within the plate wells Detection may be
accomplished using AntiMouse IgGPeroxidase
Catalog Number A9044 or equivalent diluted 110000
followed by an appropriate substrate for visualization
SigmaAldrich also offers the ANTIFLAG High
Sensitivity M2 coated 96well plate Catalog Number
P2983 for EIAbased screening applications
References
1 Brizzard BL et al Immunoaffinity purification of
FLAG epitopetagged bacterial alkaline
phosphatase using a novel monoclonal antibody
and peptide elution BioTechniques 16 730735
(1994)
2 Bjerrum OJ and Heegaard NHH CRC
Handbook of Immunoblotting of Proteins Volume I
Technical Descriptions CRC Press (Boca Raton
FL 1988) p 229236
3 Dunbar BS (ed) Protein Blotting A Practical
Approach IRL Press (New York NY 1994)
p 6770
4 Fortin A et al A 56 to 54kilodalton non grata
signal in immunoblot analysis using the horseradish
peroxidase chemiluminescence system Biochem
Cell Biol 72 239243 (1994)
5 Harlow E and Lane D Antibodies A Laboratory
Manual Cold Spring Harbor Laboratory (Cold
Spring Harbor NY 1988) Catalog No A2926
6 Ciaccia AV and Price EM IBI FLAG Epitope 1
45 (1992)
BioMax is a registered trademark of Carestream
Health Inc
FLAG and ANTIFLAG are registered trademarks and
Ezview and FLAGBAP are trademarks of Sigma
Aldrich Co LLC
Immobilon is a registered trademark of Millipore Corp
TRITON is a trademark of Dow Chemical Co
TWEEN is a registered trademark of Uniqema
Americas LLC
DABCO is a registered trademark of Air Products &
Chemicals Inc
JJPHC 03121
4
Troubleshooting Guide (Western Blot Immunostaining Procedure)
Problem Possible Cause Solution
Protein is not
expressed
Verify nucleic acid sequence and reading frame of the FLAG fusion protein in
vector construct If sequence is present attempt to optimize expression
Target protein poorly
represented in
sample
Positive controls (10 nglane recommended) should always be included If the
positive control works the sample may not contain the FLAG fusion protein of
interest or it may be present at concentrations too low to detect Immuno
precipitation with ANTIFLAG M2 Affinity Gel Catalog Number A2220 may be
required for low concentrations of FLAG fusion proteins
Positive controls available from Sigma
· Aminoterminal FLAGBAPä Fusion Protein Catalog Number P7582
· Carboxyterminal FLAGBAP Fusion Protein Catalog Number P7457
· Aminoterminal MetFLAGBAP Fusion Protein Catalog Number P5975
Defective detection
reagents
Run appropriate controls to ensure performance Use 10 nglane of a control
FLAGBAPfusion protein as a positive control If no signal is obtained with the
control repeat the procedure using a fresh lot of secondary antibodyHRP
conjugate along with freshly prepared reagents
Inadequate exposure
time using chemi
luminescent system
If no signal is observed on the film expose for longer times It is
recommended to try exposure times ranging from about 5 seconds to as long
as 10 minutes
Inappropriate film Switch to film designated for chemiluminescent detection such as BioMax
Light Catalog Number Z373494
No target protein
present on
membrane
Verify transfer onto the membrane by visualizing proteins using Ponceau S
solution Catalog Number P7170 If possible a positive control should always
be run to insure that the detection system components are functioning
normally Prestained protein markers eg Catalog Numbers C1992 or C4861
may also be used to verify complete transfer of proteins from gel to
membrane
Antigen is covered by
blocking reagent due
to overblocking
Masking of a signal can occur if the blocking reagent (such as casein or
gelatin containing blocking buffers) is used at an excessively high
concentration A dilution ranging from 11 to 13 may be performed to
decrease the concentration of blocking reagent If the problem persists use
TBS with 3 nonfat Milk Catalog Number T8793
Fusion protein is not
detected
Antibody
concentration is not
optimal
Determine the optimal working dilution for the Monoclonal ANTIFLAG M2
antibody via titration Consider using a higher concentration of antibody if no
signal or a weak signal is detected Also antibody used at an excessively high
concentration can cause signal inhibition especially in chemiluminescent
detection systems
Cellular extract
concentration is too
high
25–10 mg of total lysate protein per lane is usually enough to obtain a good
signal Load less cellular extract or serially dilute the cellular extract to
determine the optimal signal to noise ratio
Monoclonal ANTI
FLAG M2 antibody
concentration is too
high
Dilute the Monoclonal ANTIFLAG M2 antibody to a concentration ranging
from 01–05 mgml Use TBS with 3 nonfat milk as the diluent
Secondary antibody
crossreactivity
For the secondary antibody it is recommended that users initially employ
dilutions of 130000 Higher dilutions may be necessary or a more specific
secondary antibody should be used
High nonspecific
background
Monoclonal ANTI
FLAG M2 antibody
crossreacts with
naturally occurring
FLAGlike epitopes
Increasing the temperature to 37 °C during the blocking binding and wash
steps may reduce crossreactivity Lysates from mocktransfected controls
(transfected with plasmid lacking insert DNA) will help distinguish the FLAG
fusion proteins from other crossreacting proteins
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particular use Additional terms and conditions may apply Please see product information on the SigmaAldrich website at
wwwsigmaaldrichcom andor on the reverse side of the invoice or packing slip

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