Invitrogen™ GFP Monoclonal Antibody (5F12.4), eFluor™ 660, eBioscience™

Catalog No.	Quantity
50-112-4420	100 μg

Catalog No. 50-112-4420 Supplier Invitrogen™ Supplier No. 50649882

Description

Rat Monoclonal Antibody

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 flow cytometric analysis of GFP expressing 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. eFluor™ 660 is a replacement for Alexa Fluor™ 647. eFluor™ 660 emits at 659 nm and is excited with the red laser (633 nm). Please make sure that your instrument is...

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.

Specifications

• Antigen: GFP
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: 5F12.4
• Concentration: 0.2 mg/mL
• Conjugate: eFluor 660
• Formulation: PBS with 0.09% sodium azide; pH 7.2
• Gene Alias: GFP; GFP tag; GFP2; green fluorescence; green fluorescent; green fluorescent protein; Turbo eGFP
• Host Species: Rat
• Purification Method: Affinity chromatography
• Quantity: 100 μg
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Target Species: Tag
• Content And Storage: 4°C, store in dark, DO NOT FREEZE!
• Product Type: Antibody
• Form: Liquid
• Isotype: IgG2a κ
• Target Molecule: Tag

Frequently Asked Questions (FAQs)

I am unable to analyze my cells stained with eFluor Organic Dyes today. What options do I have?

Our options will depend on the samples you are analyzing.

If cell viability is not critical, you can store your stained samples at 4 degrees C or on ice overnight in the dark and analyze the following day.

For samples stained with eFluor organic fluorochromes, we recommend that cells be suspended in 100 uL of Flow Cytometry Staining Buffer (Cat. No. 00-4222) and 100 uL of eBioscience IC Fixation Buffer (Cat. No. 00-8222); samples can be incubated for up to 3 days at 4 degrees C in the dark. Alternatively, the 1-step Fix/Lyse Solution (Cat. No. 00-5333) can be used. This is a great option when working with whole blood but also works for other cell types.

Can the eFluor Organic fluorochromes be used for intracellular staining?

Yes, the eFluor Organic fluorochromes can be used for intracellular staining. The eFluor organic fluorochromes maintain bright signal and require minimal changes in compensation when fixed with eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) and Permeabilization Buffer (Cat. No. 00-8333-56) or 1-step Fix/Lyse Solution (Cat. No. 00-5333-54, 00-5333-57) (as compared to live cells).

Is the eFluor 660 fluorochrome compatible with Anti-Cy5/Alexa Fluor 647 beads?

Yes, in-house studies have demonstrated that the eFluor 660 fluorochrome is recognized by Anti-Cy5/Alexa Fluor 647 beads. Side by side studies with Alexa Fluor 647 versus eFluor 660 conjugated antibodies have demonstrated comparable results.

What buffers are compatible with the eFluor Organic fluorochromes and eVolve QDots?

The eFluor Organic fluorochromes and eVolve QDots can be used with flow staining buffers containing PBS and protein.

What is the difference between eFluor Organic Dyes and eVolve Qdots?

The eFluor Organic Dyes (eFluor 450, APC-eFluor 780, PerCP-eFluor 710, eFluor 710) are conventional fluorochromes. In contrast, the eVolve line of products are Quantum dots.

Are the eFluor Organic Dyes photo-labile?

As with other fluorochromes, we recommend minimal exposure to light to maintain optimal signal.