293T cells transiently transfected with a GFP-expressing construct were fixed using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00) and stained intracellularly with Rat IgG2a K Isotype Control PerCP-eFluor® 710 (Product # 46-4321-82) (left) or 0.06 µg of Anti-GFP PerCP-eFluor® 710 (right). Total viable cells were used for analysis.

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Product Details

46-6498-80

Applications

Tested Dilution

Publications

Flow Cytometry (Flow)

0.125 µg/test

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Product Specifications

Species Reactivity

Tag

Published species

Tag

Host/Isotype

Rat / IgG2a, kappa

Recommended Isotype Control

Rat IgG2a kappa Isotype Control (eBR2a), PerCP-eFluor™ 710, eBioscience™

Class

Monoclonal

Type

Antibody

Clone

5F12.4

Conjugate

PerCP-eFluor™ 710 PerCP-eFluor™ 710 PerCP-eFluor™ 710

Excitation/Emission Max

482/708 nm View spectra

Form

Liquid

Concentration

0.2 mg/mL

25 µg

Purification

Affinity chromatography

Storage buffer

PBS, pH 7.2

Contains

0.09% sodium azide

Storage conditions

4°C, store in dark, DO NOT FREEZE!

Shipping conditions

Ambient (domestic); Wet ice (international)

AB_11042700

Product Specific Information

Description: This 5F12.4 monoclonal antibody reacts with green-fluorescent protein (GFP), which was originally isolated from the cnidarian Aequorea victoria. This protein absorbs blue light (maximally at 395 nm) and emits green light (peak at 509) without the requirement of exogenous substrates and cofactors. These unique qualities allow GFP to be used to monitor gene expression and protein localization in vivo. Several mutant forms of GFP have been developed which fluoresce more intensely and have shifted excitation maxima when compared to wildtype GFP, making them useful for flow cytometry, fluorescence microscopy, and double-labeling applications. This antibody is capable of staining formaldehyde fixed cells.

Applications Reported: This 5F12.4 antibody has been reported for use in flow cytometric analysis.

Applications Tested: This 5F12.4 antibody has been tested by intracellular staining and flow cytometric analysis of GFP transfected cells. This can be used at less than or equal to 0.125 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.

PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.

Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-822-49) (100 µL cell sample + 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333-54) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.

Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.

Filtration: 0.2 µm post-manufacturing filtered.

Target Information

The jellyfish Aequorea victoria contains green fluorescent protein (GFP Tag) that emits light in the bioluminescence reaction of the animal. GFP has been used widely as a reporter protein for gene expression in eukaryotic and prokaryotic organisms, and as a protein tag in cell culture and in multicellular organisms. As a fusion tag, GFP can be used to localize proteins, to study their movement or to research the dynamics of the subcellular compartments where these proteins are targeted. GFP technology has revealed considerable new insights in the physiological activities of living cells. GFP is a 27 kDa monomeric protein, which autocatalytically forms a fluorescent pigment. The wild type protein absorbs blue light (maximally at 395nm) and emits green light (peak emission 508nm) in the absence of additional proteins, substrates, or co-factors. GFP fluorescence is stable, species independent and is suitable for a variety of applications. GFP has been used extensively as a fluorescent tag to monitor gene expressin and protein localization. Moreover, other applications for GFP include its use in assessing protein protein interactions in the yeast two hybrid system, and in measuring distances between proteins in fluorescence energy transfer (FRET) experiments.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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Bioinformatics

Protein Aliases: GFP; GFP tag; GFP2; green fluorescence; green fluorescent; Turbo eGFP

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GFP Monoclonal Antibody (5F12.4), PerCP-eFluor™ 710, eBioscience™

GFP Antibody (46-6498-80) in Flow