Blot flag in scii

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How to blot flag in scii

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How to cleave a flag tag?

The Flag-tag contains 8 amino acids (aa) and has the sequence motif NH2-DYKDDDDK-COOH (Asp-Tyr-Lys-Asp-. Asp-Asp-Asp-Lys). The sequence contains an enterokinase cleavage site (DDDDK). Cleavage by enterokinase removes the Flag-tag from the Flag-tagged protein.

How to remove flag tag from protein?

Enterokinase (Recombinant) Enterokinase is highly specific serine protease that hydrolysis peptide bond at the carboxyl side of lysine residue preceded by four aspartic acids (FLAG-tag). Enterokinase is used for removal of Flag-Tag from fusion proteins with the FLAG-tag.

What is the FLAG-tag in Western blot?

The Flag-tag is either N- or C-terminally cloned to the protein of interest. After expression, the recombinant Flag-tag fusion protein can be analyzed by means of ELISA, flow cytometry, immunofluorescence (IF), immunoprecipitation (IP) co-immunoprecipitation (Co-IP), protein purification, and Western blotting (WB).

How do you elute his tagged proteins?

Elution and recovery of captured His-tagged protein from an IMAC column is accomplished by using a high concentration of imidazole (at least 200 mM), low pH (e.g., 0.1 M glycine-HCl, pH 2.5) or an excess of strong chelators (e.g., EDTA). Imidazole is the most common elution agent.

How to dissolve flag peptide?

Preparation Instructions To prepare a stock solution, dissolve in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) at a concentration of 5 mg/ml. Aliquot and store at -20 C. Repeated freezing and thawing is not recommended.

What is an anti flag antibody?

Anti-FLAG antibodies are reagents that can be used to detect or stain Flag-tagged recombinant proteins. The FLAG epitope tag, is an octapeptide containing the amino acid sequence DYKDDDDK. To search for specific applications, reactivities or host species simply check the filter boxes in the left column.

How do you elute a FLAG-tag?

Elution of FLAG-tagged proteins and associated complexes can be performed either with 4 Laemmli buffer or the FLAG-antigenic peptide followed by heating at 75C.

What is flag in immunoprecipitation?

Flag-tag (or DYKDDDDK-tag) is a commonly used short peptide tag for multiple applications such as immunoprecipitation (IP), protein purification, immunofluorescence, and Western blotting (WB). In this blog, we provide an introduction to the IP of Flag-tagged proteins from cellular extracts.

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