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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About This Item
NACRES:
NA.32
UNSPSC Code:
12352203
Conjugate:
agarose conjugate
Clone:
M2, monoclonal
Application:
IP, affinity chromatography
Citations:
2532
Pricing and availability is not currently available.
conjugate
agarose conjugate
Quality Segment
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
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
Application
Anti-FLAG® M2 affinity gel has been used for western blotting, immunoprecipitation and for the purification of FLAG fusion proteins.
Learn more product details in our FLAG® application portal.
Learn more product details in our FLAG® application portal.
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
Disclaimer
FLAG® tag, 3x FLAG®, DYKDDDDK tag
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This Item | |||
|---|---|---|---|
| description purified immunoglobulin, buffered aqueous glycerol solution | description clone M2 | description 1.0-1.2 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4) | description clone M2, purified immunoglobulin, buffered aqueous glycerol solution |
| conjugate agarose conjugate | conjugate - | conjugate unconjugated | conjugate alkaline phosphatase conjugate |
| antibody form purified immunoglobulin | antibody form - | antibody form affinity isolated antibody | antibody form purified immunoglobulin |
| clone M2, monoclonal | clone M2, monoclonal | clone M2, monoclonal | clone M2, monoclonal |
| storage temp. −20°C | storage temp. −20°C | storage temp. −20°C | storage temp. −20°C |
| form buffered aqueous glycerol solution | form - | form buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4) | form buffered aqueous glycerol solution |
| shipped in wet ice | shipped in wet ice | shipped in wet ice | shipped in wet ice |
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Storage Class
10 - Combustible liquids
flash_point_f
Not applicable
flash_point_c
Not applicable
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Find documentation for the products that you have recently purchased in the Document Library.
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How do I equilibrate the resin and how can I clean it for re-use?
1 answer-
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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?
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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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Is my lysis buffer compatible with Product No. A2220, ANTI-FLAG® M2 Affinity Gel?
1 answer-
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.
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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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What is the Department of Transportation shipping information for this product?
1 answer-
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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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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