buffered aqueous glycerol solution
proteins (FLAG® tag, 3x FLAG®, DYKDDDDK tag)
affinity chromatography: suitable (FLAG® peptide, Glycine, pH3.5, 3x FLAG® peptide)
immunoprecipitation (IP): suitable
(4% agarose bead; 45-165μm bead size)
>0.6 mg/mL, resin binding capacity (FLAG-BAP)
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10 - Combustible liquids
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.
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).
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.
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.
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.
If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.
The lot specific COA document can be found by entering the lot number above under the "Documents" section.
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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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Protocol for immunoprecipitation (IP) of FLAG fusion proteins using M2 monoclonal antibody 4% agarose affinity gels
Protein purification techniques, reagents, and protocols for purifying recombinant proteins using methods including, ion-exchange, size-exclusion, and protein affinity chromatography.