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.
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.
|Product Includes||Volume||Solution Color|
|GFP Rabbit Antibody Coated Microwells||96 tests|
|GFP Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|ELISA Sample Diluent||25 ml||Blue|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|Cell Lysis Buffer (10X) 9803||15 ml|
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.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
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.
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.
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PathScan is a trademark of Cell Signaling Technology, Inc.