Detailed equilibration and regeneration protocols for this product are provided in the Product Information Sheet:
http://sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/288/183/a2220bul-ms.pdf
A2220
ANTI-FLAG® M2 Affinity Gel
purified immunoglobulin, buffered aqueous glycerol solution
ANTI-FLAG® M2 Affinity Gel
Synonym(s):
Monoclonal ANTI-FLAG® M2, ANTI-FLAG® M2 Affinity Agarose Gel, Anti-ddddk, Anti-dykddddk
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Select a Size
1 ML
$950.00
5 ML
$2,970.00
10 ML
$5,550.00
25 ML
$11,710.00
2 X 25 ML
$18,290.00
4 X 25 ML
$35,340.00
About This Item
NACRES:
NA.32
UNSPSC Code:
12352203
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Product Name
ANTI-FLAG® M2 Affinity Gel, purified immunoglobulin, buffered aqueous glycerol solution
conjugate
agarose conjugate
antibody form
purified immunoglobulin
antibody product type
primary antibodies
clone
M2, monoclonal
form
buffered aqueous glycerol solution
analyte chemical class(es)
proteins
technique(s)
affinity chromatography: suitable
immunoprecipitation (IP): suitable
matrix
(4% agarose bead; 45-165μm bead size)
isotype
IgG1
capacity
>0.6 mg/mL, resin binding capacity (FLAG-BAP)
shipped in
wet ice
storage temp.
−20°C
Quality Level
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1 of 4
This Item | F1804 | A4596 | F3165 |
|---|---|---|---|
| antibody form purified immunoglobulin | antibody form affinity isolated antibody | antibody form - | antibody form purified immunoglobulin (Purified IgG1 subclass) |
| conjugate agarose conjugate | conjugate unconjugated | conjugate agarose conjugate | conjugate unconjugated |
| clone M2, monoclonal | clone M2, monoclonal | clone - | clone M2, monoclonal |
| shipped in wet ice | shipped in wet ice | shipped in - | shipped in dry ice |
| storage temp. −20°C | storage temp. −20°C | storage temp. −20°C | storage temp. −20°C |
| technique(s) affinity chromatography: suitable, immunoprecipitation (IP): suitable | technique(s) - | technique(s) - | technique(s) western blot: 10 μg/mL (Protein A) |
Application
Anti-FLAG® M2 affinity gel has been used for western blotting, immunoprecipitation and for the purification of FLAG fusion proteins.[1][2][3]
Learn more product details in our FLAG® application portal.
Learn more product details in our FLAG® application portal.
Disclaimer
FLAG® tag, 3x FLAG®, DYKDDDDK tag
General description
Anti-FLAG M2 Affinity gel is a mouse monoclonal antibody that is covalently attached to agarose. The antibody binds FLAG at the N-terminal, Met-N-terminal, C-terminal and internal locations of fusion proteins. Binding is calcium-independent.
Elution - FLAG® peptide, Glycine, pH 3.5, 3x FLAG® peptide
Elution - FLAG® peptide, Glycine, pH 3.5, 3x FLAG® peptide
FLAG® peptide, Glycine, pH3.5, 3x FLAG® peptide
Immunogen
DYKDDDDK
Physical form
Suspension in buffered saline containing azide as preservative and 50% glycerol
Legal Information
ANTI-FLAG is a registered trademark of Merck KGaA, Darmstadt, Germany
FLAG is a registered trademark of Merck KGaA, Darmstadt, Germany
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Product No.
Description
Pricing
Storage Class
10 - Combustible liquids
wgk
WGK 1
flash_point_f
Not applicable
flash_point_c
Not applicable
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Yu Ti Cheng et al.
Proceedings of the National Academy of Sciences of the United States of America, 108(35), 14694-14699 (2011-08-30)
The nucleotide-binding domain and leucine-rich repeats containing proteins (NLRs) serve as immune receptors in both plants and animals. Overaccumulation of NLRs often leads to autoimmune responses, suggesting that the levels of these immune receptors must be tightly controlled. However, the
Michelle F Green et al.
The Journal of biological chemistry, 286(32), 28066-28079 (2011-06-15)
Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a serine/threonine-directed kinase that is activated following increases in intracellular Ca(2+). CaMKKβ activates Ca(2+)/calmodulin-dependent protein kinase I, Ca(2+)/calmodulin-dependent protein kinase IV, and the AMP-dependent protein kinase in a number of physiological pathways, including
Nora Nonne et al.
Nucleic acids research, 38(4), e20-e20 (2009-12-04)
MicroRNAs (miRNAs) bind to Argonaute proteins, and together they form the RISC complex and regulate target mRNA translation and/or stability. Identification of mRNA targets is key to deciphering the physiological functions and mode of action of miRNAs. In mammals, miRNAs
Joshua M Baughman et al.
Nature, 476(7360), 341-345 (2011-06-21)
Mitochondria from diverse organisms are capable of transporting large amounts of Ca(2+) via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter. Although the uniporter's biophysical properties have been studied extensively, its molecular composition remains elusive. We recently used comparative proteomics to
Chenggong Ji et al.
PLoS pathogens, 15(6), e1007876-e1007876 (2019-06-20)
The guanylate-binding proteins (GBPs) belong to the dynamin superfamily of GTPases and function in cell-autonomous defense against intracellular pathogens. IpaH9.8, an E3 ligase from the pathogenic bacterium Shigella flexneri, ubiquitinates a subset of GBPs and leads to their proteasomal degradation.
Articles
The FLAG® Expression System is a proven method to express, purify and detect recombinant fusion proteins. Sigma®, the proven provider of FLAG®, now offers a magnetic bead for immunoprecipitation, protein purification, and the study of protein-protein interactions. The ANTI-FLAG® M2 Magnetic Bead is composed of murine derived, anti-FLAG® M2 monoclonal antibody attached to superparamagnetic iron impregated 4% agarose beads, with an average diameter of 50 µm. The M2 antibody is capable of binding to fusion proteins containing a FLAG peptide sequence at the N-terminus, Met-N-terminus, or C-terminus locations in mammalian, bacterial, and plant extracts.
Protocols
Protocol for immunoprecipitation (IP) of FLAG fusion proteins using M2 monoclonal antibody 4% agarose affinity gels
Related Content
Protein purification techniques, reagents, and protocols for purifying recombinant proteins using methods including, ion-exchange, size-exclusion, and protein affinity chromatography.
Find protein research tools to prepare, isolate, and analyze proteins. Organized by how to extract, protect, purify, enrich, modify, and quantify proteins.
EZviewTM Red Protein A and ANTI-FLAG® M2 Affinity Gels: Immunoprecipitation with Enhanced Visibility Affinity Beads - Technical Article - July 2001
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How do I equilibrate the resin and how can I clean it for re-use?
1 answer-
Helpful?
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May I ask a question about the product? Does "1mL" mean 1 mL Slurry plus 50% glycerol, which is 2 mL in total? Same as other (e.g., 10 mL = 50% slurry with total 20 mL volume)
1 answer-
The "1mL" indicates that this product is supplied at a volume of 1 milliliter. This ANTI-FLAG M2 affinity gel is supplied as a 50% suspension in 50% glycerol with 10 mM sodium phosphate and 150 mM sodium chloride, pH 7.4, containing 0.02% (w/v) sodium azide (PBS/A).
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What is the bead size for this product?
1 answer-
As stated in the 'PROPERTIES' section of the product page, the bead size range is 45-165 μm.
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Is there a generic agarose bead available for purchase that can be used as a control to test for non-specific binding, in the case that something is binding to the agarose bead itself rather than the tag? If it is not listed on the site, is there a possibility of obtaining generic agarose beads through alternative means? Alternatively, could any information be provided to determine if another provider's generic agarose beads are produced in a similar manner for this specific purpose?
1 answer-
Yes, there are two options available for a generic agarose bead as a control to test for non-specific binding. One option is Product Number. 4B200, Sepharose® 4B, which can be used for sample preclearing and is equivalent to the beads in the FLAG affinity product. The other option is Product Number: A0919, Mouse IgG−Agarose, which can serve as a conjugated antibody bead control.
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Is there a protocol for regeneration?
1 answer-
Please see the link below to the product data-sheet. Instructions state that the column should be regenerated immediately after use by washing with three column volumes of 0.1 M glycine HCI, pH 3.5. The column should be immediately re-equilibrated in TBS until the effluent is at neutral pH:
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/288/183/a2220bul-ms.pdf
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I am using Product No. A2220, ANTI-FLAG® M2 Affinity Gel, and have a lot of non-specific proteins that are eluting with my FLAG®-tagged protein. How can I get rid of these?
1 answer-
The product bulletin for Product A2220, ANTI-FLAG® M2 affinity gel indicates:Pre-clear lysate with Mouse IgG-Agarose (Product A0919) to remove nonspecific binding proteins. Alternatively, you can use the unconjugated resin (Product 4B200) for this purpose. Other methods to remove non-specific binding from the resin would be to increase the stringency of the washes by increasing salt concentration (the resin can tolerate up to 1M NaCl) or including detergents that are compatible with the resin.
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When using Product No. A2220, ANTI-FLAG® M2 Affinity Gel, I see bands at 20-25 kDa and 50-60 kDa appearing in my Westerns that are not my FLAG®-tagged protein. How can I prevent this?
1 answer-
As a result of the conjugation, there may be some M2 antibody that is not conjugated to the resin, but is associated with the resin and may appear in acid elutions as heavy and light chain when using the anti-mouse IgG conjugated secondary antibody. We recommend an acid wash (0.1 M glycine-HCL pH 3.5) and neutralization of the resin (do not allow the acid wash to sit on the resin longer than 20 minutes) prior to applying the lysate. Another way to avoid this is to use a directly conjugated FLAG® antibody for detection such as product A8592 ANTI-FLAG® M2 HRP, or the rabbit anti-FLAG® polyclonal antibody, F7425.
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What is the Department of Transportation shipping information for this product?
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Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.
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When using ANTI-FLAG M2 Affinity Gel, Product A2220, should I use a 3X FLAG peptide or a 1X FLAG peptide to elute my protein?
1 answer-
If you have a 3X FLAG-tagged protein, then you will need to use the 3X FLAG peptide. If you have a 1X FLAG-tagged protein, you can use the 1X FLAG peptide or the 3X FLAG peptide. We have not noticed a significant difference in elution efficiency by using a 3X FLAG peptide on a 1X FLAG-tagged protein.
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How should I elute my protein when using Product No. A2220, ANTI-FLAG® M2 Affinity Gel?
1 answer-
Elution with the peptide is the most gentle method. Acid elution (0.1 M glycine-HCL pH 3.5) is a more stringent method of elution, and should be evaluated for its effect on your protein if it is to be used in downstream applications. Boiling the resin in sample buffer is the most denaturing condition. If this condition is used, the resin cannot be re-used, due to the presence of SDS and/or reducing agents. The elution information can be viewed on A2220 product information sheet (under Documents, above).
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