Green Fluorescent Protein (GFP) antibody

Antigen: Green Fluorescent Protein (GFP)

Clonality: Polyclonal

Host: Chicken

Application: ELISA, Western Blotting (WB), Immunofluorescence (IF)

Catalog no.: ABIN349609

Additional Information

Alternative name: Green Fluorescent Protein (Aequorea victoria)

Immunogen: The immunogen is a Green Fluorescent Protein (GFP) fusion protein corresponding to the full length amino acid sequence (246aa) derived from the jellyfish Aequorea victoria.

Format: Liquid (sterile filtered)

Specificity: This product was prepared from egg yolks by immunoaffinity chromatography using Green Fluorescent Protein (Aequorea victoria) coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities. Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Chicken Serum and purified and partially purified Green Fluorescent Protein (Aequorea victoria). No reaction was observed against Human, Mouse or Rat serum proteins.

Application Details

Application Notes: ELISA:1:2,000 - 1:12,000 WB:1:200 - 1:1,000 IHC:User Optimized OTHER: User Optimized Polyclonal anti-GFP is designed to detect GFP and its variants. This antibody can be used to detect GFP by ELISA (sandwich or capture) for the direct binding of antigen and recognizes wild type, recombinant and enhanced forms of GFP. Biotin conjugated polyclonal anti-GFP used in a sandwich ELISA is well suited to titrate GFP in solution using this antibody in combination with Rockland's monoclonal anti-GFP (600-301-215) using either form of the antibody as the capture or detection antibodies. However, use the monoclonal form only for the detection of wild type or recombinant GFP as this form does not sufficiently detect 'enhanced' GFP. The detection antibody is typically conjugated to biotin and subsequently reacted with streptavidin conjugated HRP (code # S000-03). Fluorochrome conjugated polyclonal anti-GFP can be used to detect GFP by immunofluor-escence microscopy in prokaryotic (E.coli) and eukaryotic (CHO cells) expression systems and can detect GFP containing inserts. Significant amplification of signal is achieved using fluorochrome conjugated polyclonal anti-GFP relative to the fluorescence of GFP alone. For immunoblotting use either alkaline phosphatase or peroxidase conjugated polyclonal anti-GFP to detect GFP or GFP containing proteins on western blots.

Concentration: 0.64 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 or below prior to opening. Dilute only prior to immediate use. For extended storage, aliquot contents and freeze at -20°C or below. Avoid cycles of freezing and thawing. Expiration date is one (1) year from date of opening product.

Research Area: Tags/Labels

Restrictions: For Research Use only

Images

Western blot of GFP protein detected with antibodies-online's polyclonal anti-GFP antibody. Lane 1 shows negative control staining of 20 ug of mouse spleen lysate. Lane 2 shows staining of mouse spleen lysate spiked with 50 ng of wt GFP. This antibody detects a 27 kDa band corresponding to the GFP epitope tag commonly used in recombinant constructs. A 4-20% Tris-Glycine gradient gel was used for SDS-PAGE followed by transfer to nitrocellulose using standard methods. After blocking with 5% BSA in PBS, the membrane was probed for 2 h at room temperature with the primary antibody diluted in 5% BSA to 2 ug/ml, followed by washes and reaction with a 1:20,000 dilution of IRDye TM 800 Conjugated Affinity Purified anti-Chicken IgG [H&L] [Goat] MX10 (p/n 603-132-126) . The IRDye 800 fluorescence image was captured using the Odyssey Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.

Immunofluorescence microscopy. Chicken-anti-GFP was used to detect a recombinant tau-myc-GFP protein expressed in situ under the control of the KruppleGAL4 driver line in Drosophila eye disc. Detection occurred using the anti-GFP antibody diluted 1:500 (green) for 2 h at RT followed by a 1:300 dilution of AlexaTM488 conjugated anti-Chicken IgG for 1 h at RT. Blocking occurred using 5% NGS in PBS with 0.1% Triton X-100 for 15 min. In separate experiments, endogenous GFP was not detectable after fixation without antibody staining (data not shown). Images were collected using a Leica TCS SPII confocal imaging system. Personal Communication, Haluk Lacin, Developmental Biology Program and Jim Skeath, Genetics Department, Washington University School of Medicine, St. Louis, MO.