GFP Quantitation Kit

Catalog No. ABIN2344797

Product Details

Detection Method: Fluorometric

Application: Quantification (Q)

Sample Type: Tissue Samples, Cell Samples

Characteristics: GFP Quantitation Kit measures GFP fluorescence in a fluorometer. The quantity of rGFP in sample is determined by comparing its fluorescence reading with that of known recombinant GFP standard curve. The kit has detection sensitivity limit of 100 ng/mL. A proprietary GFP quench solution is also included for determining autofluorescence of cell or tissue sample. Each kit provides sufficient reagents to perform up to 100 assays including standard curve and GFP samples.

Components:

1. 5X Assay/Lysis Buffer : One 30 mL bottle
2. Assay Diluent : One 30 mL bottle
3. 10X GFP Quench Solution : One 12 mL bottle.
4. GFP Standard : One 20 μL vial of purified recombinant GFP at 1 mg/mL in 1X PBS

Material not included:

1. Cells or tissues expressing GFP or GFP fusion protein
2. Heating block
3. 96-well plate suitable for a fluorescence plate reader
4. Fluorescence plate reader

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Convenient direct fluorescence measurement
• Simpler and faster than FACS analysis

Reagent Preparation:

• 1X Assay/Lysis Buffer: Mix the 5X Stock briefly and dilute to 1X in deionized water. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin.
• 10X GFP Quench Solution: If precipitated crystals are present, briefly heat the solution at 37 °C to redissolve the crystals.

Assay Procedure:

1. Prepare cell or tissue lysates containing GFP or GFP fusion protein in 1X Assay/Lysis Buffer.
2. Transfer 100 μL of the GFP lysate sample to a 96-well plate suitable for fluorescence measurement. Read the fluorescence with a fluorescence plate reader at 488 nm/507 nm. Each GFP sample, blank, and control lysate should be assayed in duplicate. Autofluorescence Measurement (optional)
   1. Transfer 180 μL of the GFP sample to an eppendorf tube. Add 20 μL of 10X GFP Quench Solution. In a heating block, incubate the sample at 65 °C for 15 minutes.
   2. Cool the solution to 37 °C or room temperature in a water bath for 5 minutes. 3
   3. Centrifuge 5 minutes at 10,000 g and transfer 100 μL of the supernatant to a 96-well plate suitable for fluorescence measurement. Read the fluorescence with a fluorescence plate reader at 488 nm/507 nm.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store all kit components at 4°C.

References

Pfeiffer, Truman, Rubin: "Using translational enhancers to increase transgene expression in Drosophila." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, Issue 17, pp. 6626-31, (2012) (PubMed).

Wamboldt, Mohammed, Elowsky, Wittgren, de Paula, Mackenzie: "Participation of leaky ribosome scanning in protein dual targeting by alternative translation initiation in higher plants." in: The Plant cell, Vol. 21, Issue 1, pp. 157-67, (2009) (PubMed).

Target Details

Background: Green Fluorescent Protein (GFP) is a spontaneously fluorescent protein originally isolated from the jellyfish Aequorea victoria. Molecular cloning of the GFP gene and its subsequent expression in heterologous systems have established recombinant GFP (rGFP) as a valuable reporter molecule for in vivo visualization of gene expression events in a wide variety of cell types and organisms. Since rGFP requires no additional substrates or cofactors, rGFP fluorescence can be easily detected under fluorescence microscope after expression in either prokaryotic or eukaryotic cells. Most imaging studies of rGFP are qualitative. Quantitative analysis of rGFP level in cells by FACS is time consuming and expensive.