Enterokinase Conjugated Proteins (Cleavage Site DYKDDDDK Tag) antibody

Antigen: Enterokinase Conjugated Proteins

Epitope: Cleavage Site DYKDDDDK Tag

Clonality: Polyclonal

Host: Rabbit

Application: ELISA, Western Blotting (WB), Immunoprecipitation (IP)

Catalog no.: ABIN99294

Additional Information

Alternative name: Enterokinase (ECS) Cleavage Site (DYKDDDDK Tag) Conjugated Proteins

Immunogen: This antibody was purified from whole rabbit serum prepared by repeated immunizations with the FLAG epitope tag peptide DYKDDDDK (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) conjugated to KLH using maleimide. Residues of glycine and cysteine were added to the carboxy terminal end to facilitate coupling.

Format: Liquid (sterile filtered)

Description: Epitope tags are short peptide sequences that are easily recognized by tag-specific antibodies. Due to their small size, epitope tags do not affect the tagged protein's biochemical properties. Most often sequences encoding the epitope tag are included with target DNA at the time of cloning to produce fusion proteins containing the epitope tag sequence. This allows anti-epitope tag antibodies to serve as universal detection reagents for any tag containing protein produced by recombinant means. This means that anti-epitope tag antibodies are a useful alternative to generating specific antibodies to identify, immunoprecipitate or immunoaffinity purify a recombinant protein. The anti-epitope tag antibody is usually functional in a variety of antibody-dependent experimental procedures. Expression vectors producing epitope tag fusion proteins are available for a variety of host expression systems including bacteria, yeast, insect and mammalian cells. Antibodies-Online Immunochemicals produces anti-epitope tag antibodies against many common epitope tags including Myc, GST, GFP, 6X His, MBP, FLAG and HA. Antibodies-Online Immunochemicals also produces antibodies to other tags including FITC, Rhodamine (TRITC), DNP and biotin.

Specificity: This affinity purified antibody is directed against the FLAG motif and is useful in determining its presence in various assays. This polyclonal anti-FLAG tag antibody detects over-expressed proteins containing the FLAG epitope tag. To date this antibody has reacted with all amino- terminal FLAG tagged proteins so far tested. In western blotting of bacterial extracts the antibody does not cross-react with endogenous proteins.

Application Details

Application Notes: Recommended Dilutions: This product was assayed by immunoblot and was found to be reactive at 1:1,000 to 1:5,000. For ELISA a dilution of 1:4,000 to 1:16,000 is suggested for detection of FLAG containing recombinant proteins or for screening in microtiter plates of recombinants. Use antibodies-online's HRP Anti-RABBIT IgG (H&L) (DONKEY) MX Bv Ch Gt GP Hs Hu Ms Rt & Sh (code 611-703-127) as a secondary antibody for detection. The researcher should determine optimal titers for other applications. Note: Anti-FLAG is optimally suited for monitoring the expression of FLAG tagged fusion proteins. As such, anti-FLAG/FLAG can be used to identify fusion proteins containing the FLAG epitope. The antibody recognizes the FLAG epitope tag fused to the amino- terminus of targeted proteins. No reactivity is seen against fusion proteins containing the FLAG epitope tag at the carboxy- terminus. This antibody has been tested by ELISA and western blotting against both the immunizing peptide and FLAG containing recombinant proteins. Although not tested, this antibody is likely functional for immunoprecipitation and immunocytochemistry, and other immunodetection techniques. The FLAG epitope tag peptide sequence was first derived from the 11-amino-acid leader peptide of the gene-10 product from bacteriophage T7. Now the most commonly used hydrophilic octapeptide is DYKDDDDK. Antibodies-Online Immuno-chemical's polyclonal anti-FLAG epitope tag antibody binds FLAG containing fusion proteins with greater affinity than the widely used monoclonal M1, M2 and M5 clones, and shows greater sensitivity in most assays. Affinity purification of the polyclonal antibody results in very low background levels in assays and low cross-reactivity with other cellular proteins. 2.

Concentration: 0.27 mg/ml (by UV absorbance at 280 nm)

Buffer: Stabilizer: None. Preservative: 0.01% (w/v) Sodium Azide. Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2.

Storage: Store vial at -20° C. For extended storage aliquot contents and freeze at -20° C or below. Avoid cycles of freezing and thawing. Dilute only prior to immediate use. Expiration date is one (1) year from date of opening.

Research Area: Tags/Labels

Restrictions: For Research Use only

Images

Anti-FLAG epitope tag polyclonal antibody detects amino-terminal FLAG tagged recombinant proteins by western blot and not carboxy- terminal fusion proteins. Polyclonal rabbit-anti-FLAG epitope tag at 1:2,500 dilution was used to detect 1.0 ug of recombinant protein containing the FLAG epitope tag. Lane 1 shows reactivity against amino- terminal FLAG-Bacterial Alkaline Phosphatase (BAP) fusion protein (Sigma P-7582). Lane 2 shows no reactivity against carboxy- terminal FLAG-BAP fusion protein (Sigma P-7457). A 4-20% gradient gel was used to separate the protein by SDS-PAGE. The protein was transferred to nitrocellulose using standard methods. After blocking the membrane was probed with the primary antibody for 1 h at room temperature followed by washes and reaction with a 1:5,000 dilution of IRDye

Publications

Publications: Urano, Ichikawa, Morikawa et al.: "T cell-based functional cDNA library screening identified SEC14-like 1a carboxy-terminal domain as a negative regulator of human immunodeficiency virus replication." in: Vaccine, Vol. 28 Suppl 2, pp. B68-74, 2010 (PubMed).