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Cat. No.: AFAG2-0.5 (for 0.5ml)

Cat. No.: AFAG2-2 (for 2ml)

Cat. No.: AFAG2-10 (for 10ml)

Description

Anti-Flag Affinity Gel v2 is made by covalently coupling high-quality mouse-derived Flag antibodies to agarose. It binds to proteins with Flag tags in standard protein expression systems (such as bacteria, yeast, or mammalian cells), making it suitable for the purification or immunoprecipitation (IP) of Flag-tagged fusion proteins or protein complexes.

Flag tags (Flag-tag), Myc tags (Myc-tag), HA tags (HA-tag), His tags (His-tag), and GST tags (GST-tag) are common tags on commercial expression vectors. These tags facilitate the detection of target protein expression and functionality, or the purification of fusion proteins. The Flag tag is an 8-amino-acid peptide (DYKDDDDK) and is often used in forms such as Flag and 3X Flag. The Flag-tag sequence can be genetically fused to either the N-terminus or C-terminus of the target gene.

The advantages of the Flag tag include:

• It generally does not interact with the target protein or affect its function and properties, facilitating downstream studies of fusion proteins.
• As a tag protein, the Flag tag allows subsequent detection of gene expression, localization, and function, as well as protein purification and immunoprecipitation, using Flag antibodies or Anti-Flag Affinity Gel.
• When fused to the N-terminus, the Flag tag can be cleaved by enterokinase (EK) to yield the specific target protein.
• Due to these advantages, the Flag tag is widely used in protein expression, purification, identification, functional studies, and related protein-protein interaction research.

Anti-Flag Affinity Gel v2, also known as Anti-DYKDDDDK Affinity Gel/Beads/Resin, specifically binds to Flag fusion proteins and is widely used for purifying or immunoprecipitating Flag-tagged fusion proteins or protein complexes.

Features

• This product has a high binding capacity and strong specificity for fusion proteins: Each ml of settled gel contains approximately 8 mg of Flag antibody, capable of binding about 1 mg of fusion protein, with minimal non-specific protein binding.
• Versatile binding to various forms of Flag-tagged proteins: This product can specifically bind methionine-modified N-terminal Flag fusion proteins (Met-Flag-Protein), N-terminal Flag fusion proteins (Flag-Protein), C-terminal Flag fusion proteins (Protein-Flag), or Flag-tagged proteins with internal Flag sites.
• Multiple elution methods available: Depending on the protein's structural integrity, biological function, and subsequent applications, various elution methods can be used, including Flag peptide, acidic, alkaline, neutral, and SDS-PAGE loading buffer. Notably, elution with Flag peptide does not include antibody light and heavy chains, effectively addressing interference in Western blot assays following immunoprecipitation.
• Reusable and cost-effective: Under normal conditions, this product can be reused 4-10 times for purifying the same protein. However, it is not recommended for reuse in immunoprecipitation assays detecting protein-protein interactions.
• Easy to use: This product is stored in TBS buffer without glycerol, eliminating the need to remove glycerol before use. It is easy to mix and pipette, reducing sample loss.

• This product is compatible with various reagents: It can be used with certain concentrations of DTT, EDTA, common detergents, guanidine hydrochloride, and urea, as detailed in the table below.

  Reagent	Maximum Tolerable Concentration	Comments
  EDTA	5 mM	Higher concentration of chelating agent will reduce purification efficiency with less target protein recovery.
  β-ME, DTT	100 mM for β-ME; 80 mM for DTT	Reducing agents will reduce disulfide bonds in the G1 antibody on the gel. Avoid reducing agents or keep at low concentration (<10 mM) during purification. When reducing agents reach the maximum tolerable concentration, antibody heavy chain (~50 kDa) and light chain (~25 kDa) bands will appear in SDS-PAGE analysis. The gel CANNOT BE REUSED if samples containing higher concentrations of reducing reagents are applied to the gel.
  Tween 20, Triton X-100	5%	The concentration of Tween 20 and Triton X-100 should not exceed 5%.
  SDS	Not suggested	SDS will denature the G1 antibody on the gel.
  NP-40	4%	Higher concentration of NP-40 will reduce purification efficiency with less target protein recovery.
  GuHCl, Urea	0.3 M for GuHCl; 1.5 M for Urea	Chaotropic reagents will denature the target Flag-tagged protein. Do not exceed 0.3 M GuHCl or 1.5 M Urea.
  Glycerol	20%	Higher concentration of glycerol will interfere with the binding of Flag-tagged protein.
  NaCl	0.5 M	Higher concentration of NaCl will reduce purification efficiency with less target protein recovery.

Parameters

• Product Content: 50% settled gel in TBS with preservative
• Matrix: 4% cross-linked agarose
• Average Bead Size: 90 μm
• Antibody: Mouse monoclonal antibody against Flag-tag, clone G1
• Isotype: IgG2b
• Molecular Weight of Antibody: Approximately 150 kDa
• Antibody Concentration: Approximately 8 mg Flag antibody per ml settled gel
• Binding Capacity: Approximately 1 mg Flag-tagged protein per ml settled gel
• Gel Reuse: The gel can be recycled at least 4 times. If maintained properly, it can be reused up to 10 times with minimal loss of binding capability.
• Elution Method: Acidic, alkaline, neutral, peptide competitive, or SDS-PAGE loading buffer elution. Note: When eluted with SDS-PAGE loading buffer, the light (~25 kDa) and heavy (~50 kDa) chains of the G1 antibody will be denatured and released from the gel.
• Reagent Compatibility: Compatible with commonly used reagents at certain concentrations.
• Application: Suitable for protein purification, pull-down, IP, and Co-IP.

Storage

Store at 4°C, valid for one year.

Precautions

• This product does not contain glycerol; do not freeze.
• Ensure thorough resuspension before use by inverting several times to mix well.
• The product contains trace amounts of preservative, which should not affect routine protein or protein complex purification and immunoprecipitation. However, if enzyme activity assays are involved, wash the gel with TBS or another suitable solution three times before use to remove any potential interference from the preservative.
• When performing immunoprecipitation or purification, it is recommended to include positive and negative control groups.
• After collecting protein samples, proceed with purification promptly and keep them at 4°C or in an ice bath to minimize protein degradation. To effectively inhibit protein degradation, add an appropriate amount of protease inhibitor mix to the lysis buffer.
• If centrifugation does not completely remove insoluble matter from the protein samples, filter the sample solution with a 0.45 μm filter membrane.
• This product is for scientific research use by trained professionals only and is not intended for clinical diagnosis or treatment, food, or drug use. Do not store in residential areas.
• For your safety and health, please wear a lab coat and disposable gloves while handling.

Related:

• Anti-Flag Affinity Gel v2
• Anti-Flag Affinity Gel
• Anti-Myc Affinity Gel
• Anti-HA Affinity Gel
• Anti-V5 Affinity Gel

Only for research and not intended for treatment of humans or animals

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