A2220

Millipore

ANTI-FLAG® M2 Affinity Gel

purified immunoglobulin, buffered aqueous glycerol solution

Synonym(s):
Anti-ddddk, Anti-dykddddk, Monoclonal ANTI-FLAG M2 antibody produced in mouse, ANTI-FLAG M2 Affinity Agarose Gel
NACRES:
NA.32

Quality Level

antibody form

purified immunoglobulin

antibody product type

primary antibodies

clone

M2, monoclonal

form

buffered aqueous glycerol solution

analyte chemical class(es)

proteins (FLAG® tag, 3x FLAG®, DYKDDDDK tag)

application(s)

affinity chromatography: suitable (FLAG® peptide, Glycine, pH3.5, 3x FLAG® peptide)
immunoprecipitation (IP): suitable

matrix

(4% agarose bead; 45-165μm bead size)

isotype

IgG1

capacity

>0.6 mg/mL, resin binding capacity (FLAG-BAP)

conjugate

agarose conjugate

shipped in

wet ice

storage temp.

−20°C

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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

Immunogen

DYKDDDDK

Application

Anti-FLAG® M2 affinity gel has been used for western blotting, immunoprecipitation and for the purification of FLAG fusion proteins.

Browse additional application references in our
FLAG® Literature portal.

Physical form

Suspension in buffered saline containing azide as preservative and 50% glycerol

Legal Information

ANTI-FLAG is a registered trademark of Sigma-Aldrich Co. LLC
FLAG is a registered trademark of Sigma-Aldrich Co. LLC

RIDADR

NONH for all modes of transport

WGK Germany

WGK 1

  1. Is my lysis buffer compatible with Product No. A2220, ANTI-FLAG® M2 Affinity Gel?

    The A2220 product information sheet (under Documents, above) contains a reagent compatibility table located on page 6. We do not recommend addition of SDS or reducing agents such as DTT, DTE or 2-mercaptoethanol to the resin. If you have a detergent listed in the table in a higher than recommended concentration, we recommend trying to dilute the sample before applying to the resin.

  2. How should I elute my protein when using Product No. A2220, ANTI-FLAG® M2 Affinity Gel?

    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).

  3. 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?

    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.

  4. 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?

    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.

  5. 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?

    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. 

  6. Which document(s) contains shelf-life or expiration date information for a given product?

    If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.

  7. How do I get lot-specific information or a Certificate of Analysis?

    The lot specific COA document can be found by entering the lot number above under the "Documents" section.

  8. How do I find price and availability?

    There are several ways to find pricing and availability for our products. Once you log onto our website, you will find the price and availability displayed on the product detail page. You can contact any of our Customer Sales and Service offices to receive a quote.  USA customers:  1-800-325-3010 or view local office numbers.

  9. What is the Department of Transportation shipping information for this product?

    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. 

  10. My question is not addressed here, how can I contact Technical Service for assistance?

    Ask a Scientist here.

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...
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...
William G Roach et al.
The Biochemical journal, 403(2), 353-358 (2007-02-06)
Insulin stimulation of the trafficking of the glucose transporter GLUT4 to the plasma membrane is controlled in part by the phosphorylation of the Rab GAP (GTPase-activating protein) AS160 (also known as Tbc1d4). Considerable evidence indicates that the phosphorylation of this...
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....
Tsutomu Endo et al.
Reproduction (Cambridge, England), 141(4), 397-405 (2011-01-18)
In mammalian oocytes, histone H3 and histone H4 (H4) in the chromatin are highly acetylated at the germinal vesicle (GV) stage, and become globally deacetylated after GV breakdown (GVBD). Although nuclear core histones can be exchanged by cytoplasmic free histones...
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.
Read More
Protocols
Protocol for immunoprecipitation (IP) of FLAG fusion proteins using M2 monoclonal antibody 4% agarose affinity gels
Read More
Related Content
Protein purification techniques, reagents, and protocols for purifying recombinant proteins using methods including, ion-exchange, size-exclusion, and protein affinity chromatography.
Read More

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