Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total GFP Sandwich ELISA Kit #7878

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
GFP Ab Coated Microwells 96 tests
GFP Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
Sample Diluent 25 ml Blue
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

All

Reactivity Key: All=All species expected

Description

The PathScan® Total GFP Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects exogenously expressed GFP. This kit is intended for use with cotransfected GFP as a convenient means of monitoring transfection efficiency. A GFP rabbit antibody has been coated onto the microwells. After incubation with cell lysates, GFP is captured by the coated antibody. Following extensive washing, a GFP mouse detection antibody is added to detect the captured GFP. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. The HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of GFP.

Specificity / Sensitivity

CST's PathScan® Total GFP Sandwich ELISA Kit #7878 detects GFP when exogenously expressed in cells. As shown in Figure 1, this kit readily detects GFP in transfected, but not in untransfected HEK293 cell lysate. This kit was developed for use with transfected full-length GFP and may not detect GFP fusion proteins. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Transfected GFP is detected by PathScan® Total GFP Sandwich ELISA Kit #7878. Lysates were prepared from control and GFP-transfected HEK293 cells and analyzed for GFP by PathScan® Total GFP Sandwich ELISA Kit #7878 or by Western blotting. Absorbance readings at 450 nm are shown in the top figure while the corresponding Western blot using GFP Antibody #2555 is shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2. The relationship between the protein concentration of the lysate from untransfected and GFP-transfected HEK 293 cells and the absorbance at 450 nm is shown.

Background

The green-fluorescent protein (GFP) functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea. GFP maximally absorbs light at 395 nm and has an emission spectrum that peaks at 509 nm (1). Among other applications, GFP is commonly cotransfected alongside a gene of interest as a means of monitoring transfection efficiency.

  1. Miyawaki, A. et al. (2003) Curr Opin Chem Biol 7, 557-62.

Application References

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Protocols

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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