Luminescent Protein Imaging Without Membrane Transfer or Antibodies
Eliminate the need for immunoblotting to detect NanoLuc® fusion proteins separated by polyacrylamide gel electrophoresis. Directly image gels after incubation with the Nano-Glo® In-Gel Detection Reagent. For native PAGE, the gels can be incubated with detection reagent and imaged in less than 15 minutes. For denaturing SDS-PAGE, two washes with 25% isopropanol followed by two washes in water are needed to remove the SDS and allow the NanoLuc® luciferase to refold before detection.
The sensitivity of NanoLuc® luciferase means proteins do not need to be overexpressed to be visualized by the Nano-Glo® In-Gel Detection System.
Easily detect NanoLuc® protein fusions to:
Verify expression levels
Confirm correct molecular weight
Multiplex measurements of multiple luminescent species
Overview of the the Nano-Glo® In-Gel Detection System.
Specific and Sensitive Luminescent Detection
To highlight the ease and specificity in which NanoLuc®-tagged proteins can be detected, HEK293 cells were transiently transfected with CMV expression constructs for five different protein kinases fused to NanoLuc® luciferase. Proteins were expressed in cells both individually and in combinations as shown in the figure to the right.
Cells transfected with carrier DNA alone showed no luminescent background bands, while transfected cells showed fusion proteins of the expected molecular weights. The ability to separate the signals from different NanoLuc® fusions expressed in the same cells highlights the possibility for multiplexing the quantification of multiple proteins (e.g., comparing the levels of a regulated protein and a constitutively expressed control protein).
SDS-PAGE of transiently transfected HEK293 cells. HEK293 cells were transiently transfected individually or in combinations with five different fusions of NanoLuc® luciferase to protein kinases: NEK7, STK32A, CAMKK2, RPS6KA5 and STK10. CMV expression constructs were diluted 100-fold into carrier DNA to lower expression levels. After overnight expression, gel electrophoresis was performed using 10μl of cell lysate. Panel A. The NanoLuc® fusions were visualized using the Nano-Glo® In-Gel Detection System. The image represents a 30-second exposure immediately after adding reagent. Panel B. The gel was subsequently incubated with Coomassie® dye and imaged by transillumination to show total protein in the lysate.
原厂资料：
Luminescent Protein Imaging Without Membrane Transfer or Antibodies
Eliminate the need for immunoblotting to detect NanoLuc® fusion proteins separated by polyacrylamide gel electrophoresis. Directly image gels after incubation with the Nano-Glo® In-Gel Detection Reagent. For native PAGE, the gels can be incubated with detection reagent and imaged in less than 15 minutes. For denaturing SDS-PAGE, two washes with 25% isopropanol followed by two washes in water are needed to remove the SDS and allow the NanoLuc® luciferase to refold before detection.
The sensitivity of NanoLuc® luciferase means proteins do not need to be overexpressed to be visualized by the Nano-Glo® In-Gel Detection System.
Easily detect NanoLuc® protein fusions to:
Verify expression levels
Confirm correct molecular weight
Multiplex measurements of multiple luminescent species
Overview of the the Nano-Glo® In-Gel Detection System.
Specific and Sensitive Luminescent Detection
To highlight the ease and specificity in which NanoLuc®-tagged proteins can be detected, HEK293 cells were transiently transfected with CMV expression constructs for five different protein kinases fused to NanoLuc® luciferase. Proteins were expressed in cells both individually and in combinations as shown in the figure to the right.
Cells transfected with carrier DNA alone showed no luminescent background bands, while transfected cells showed fusion proteins of the expected molecular weights. The ability to separate the signals from different NanoLuc® fusions expressed in the same cells highlights the possibility for multiplexing the quantification of multiple proteins (e.g., comparing the levels of a regulated protein and a constitutively expressed control protein).
SDS-PAGE of transiently transfected HEK293 cells. HEK293 cells were transiently transfected individually or in combinations with five different fusions of NanoLuc® luciferase to protein kinases: NEK7, STK32A, CAMKK2, RPS6KA5 and STK10. CMV expression constructs were diluted 100-fold into carrier DNA to lower expression levels. After overnight expression, gel electrophoresis was performed using 10μl of cell lysate. Panel A. The NanoLuc® fusions were visualized using the Nano-Glo® In-Gel Detection System. The image represents a 30-second exposure immediately after adding reagent. Panel B. The gel was subsequently incubated with Coomassie® dye and imaged by transillumination to show total protein in the lysate.
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