Non-destructive analysis of cells on the molecular degree is of vital significance for cell analysis. At current, immunoassay-based and aptamer-based strategies can obtain non-structural damaging cell analysis, however nonetheless result in modifications in cells on the molecular degree. Here, we’ve proposed a dual-terminal amplification (DTA) technique, which permits nondestructive analysis of membrane protein MUC1 with out the impact on protein expression and cell viability in living cells.
Methods: A fluorophore (Cy5)-labeled DNA ternary complicated consisting of three oligonucleotides is designed. It can acknowledge MUC1 via its aptamer area, and thus make the MUC1 of cells seen beneath a fluorescence microscope.
When DNA polymerase is added, dual-terminal amplification is carried out. One path dissociates aptamer from MUC1, and the opposite path, also called rolling circle amplification (RCA), produces lengthy linear DNA strands, which could be additional adopted for quantitative analysis of MUC1. In this manner, all reagents are faraway from the floor of the cells after the analysis, which permits nondestructive analysis.
Nondestructive analysis of tumor-associated membrane protein MUC1 in living cells based on dual-terminal amplification of a DNA ternary complicated.
Description: Wnt pathway inhibitor 3 is a potent wnt inhibitor with an IC50 value of 45 nM. Wnt pathway inhibitor 3 shows antiproliferative activity[1].
Description: The AP1 Reporter Kit is designed for monitoring the activity of the JNK signaling pathway and the transcriptional activity of AP1 in cultured cells. The kit contains a transfection-ready AP1 luciferase reporter vector. This reporter contains the firefly luciferase gene under the control of multimerized AP1 responsive elements located upstream of a minimal promoter. The AP1 reporter is premixed with a constitutively-expressing Renilla (sea pansy) luciferase vector that serves as an internal control for transfection efficiency. The kit also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains the firefly luciferase gene under the control of a minimal promoter, without any additional response elements. The negative control is critical for determining pathway-specific effects and the background luciferase activity.
Description: The mammalian Target of Rapamycin (TOR, also known as mTOR) is an evolutionarily conserved serine/threonine kinase that regulates cell growth and cell cycle progression through its ability to integrate signals from nutrient levels and growth factors. TOR regulation is accomplished through a network of various activators and repressors. It is phosphorylated by Akt, whose activity is indirectly inhibited by the lipid phosphatase PTEN. TOR is normally associated with the regulatory proteins RAPTOR, a scaffold protein whose binding by TOR substrates is necessary for effective TOR-catalyzed phosphorylation, and GΒL, which stimulates TOR’s kinase activity towards downstream proteins. It is further regulated by the proteins Rheb, TSC1 and TSC2, which act to modulate TOR activity. The downstream targets of TOR are thought to be the ribosomal protein S6 kinases and the eukaryotic initiation factor 4E binding proteins (4EBPs) whose activation leads to increased protein translation and cell growth.;;For images please see PDF data sheet
Description: The FOXO Reporter kit is designed to monitor activity of the PI3K/AKT signaling pathway and the transcriptional activity of FOXO proteins in cultured cells. The kit contains the transfection-ready FOXO3 expression vector and the FOXO luciferase reporter vector, which is a PI3K/Akt pathway-responsive reporter. This reporter contains a firefly luciferase gene under the control of multimers of the FOXO responsive element located upstream of a minimal promoter. The FOXO reporter is premixed with a constitutively-expressing Renilla (sea pansy) luciferase vector, which serves as an internal control for the transfection efficiency. The kit also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as negative control. The non-inducible luciferase vector also contains the firefly luciferase gene under the control of a minimal promoter, but without any additional response elements. The negative control is critical to determining pathway-specific effects and background luciferase activity.
Description: Notch Pathway Reporter kit is designed for monitoring the activity of the Notch signaling pathway in cultured cells. The kit contains a transfection-ready expression vector for Mouse NOTCH1 that has a deletion of the entire extracellular domain and transmembrane region (NOTCH1 NICD). Inside the cells, the NOTCH1 NICD is constitutively localized into the nucleus without needing to be cleaved by γ-secretase. The kit also contains CSL (CBF1/RBP-Jκ) luciferase reporter vector, which is a Notch pathway-responsive reporter. This reporter contains the firefly luciferase gene under the control of multimerized CSL responsive elements upstream of a minimal promoter. The CSL (CBF1/RBP-Jκ) reporter is premixed with constitutively expressing Renilla (sea pansy) luciferase vector, which serves as an internal positive control for transfection efficiency._x000D_The kit also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, but without any additional response elements. The negative control is critical for determining pathway specific effects and background luciferase activity._x000D_This kit contains the expression vector for Mouse Notch1 NICD, which does not require γ-secretase processing to become active. We also offer the Mouse Notch1DE expression vector (BPS Bioscience #60509), as well as a Human Notch1DE expression vector (BPS Bioscience #79503), which are dependent on γ-secretase activity to become active.
Description: The Myc Pathway Reporter kit is designed for monitoring the activity of the Myc signaling pathway in cultured cells. The kit contains a transfection-ready expression vector for c- Myc and Myc luciferase reporter vector. Inside the cells, c-Myc will bind to Max, translocate to the nucleus, and induce expression of the Myc luciferase reporter vector. This reporter contains the firefly luciferase gene under the control of multimerized Myc responsive elements located upstream of a minimal promoter. The Myc reporter is premixed with constitutively-expressing Renilla (sea pansy) luciferase vector, which serves as an internal positive control for transfection efficiency._x000D_The kit also includes a non-inducible firefly luciferase vector premixed with constitutivelyexpressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, but without any additional response elements. The negative control is critical for determining pathway-specific effects and background luciferase activity.
Description: The ARE Reporter kit is designed for monitoring the activity of the Nrf2 antioxidant pathway in cultured cells. The kit contains a transfection-ready ARE luciferase reporter vector, which is an Nrf2 pathway-responsive reporter. This reporter contains a firefly luciferase gene under the control of multimerized ARE responsive elements located upstream of a minimal promoter. The ARE reporter is premixed with a constitutively expressing Renilla (sea pansy) luciferase vector that serves as an internal control for transfection efficiency. The kit also includes a non-inducible firefly luciferase vector premixed with constitutively expressing Renilla luciferase vector as negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, without any additional response elements. This negative control is critical to determining pathway-specific effects and background luciferase activity.
Description: The STAT3 Reporter kit is designed for monitoring the activity of the STAT3 signaling pathway in cultured cells. The kit contains transfection-ready STAT3 luciferase reporter vector. This reporter contains a firefly luciferase gene under the control of STAT3-responsive element located upstream of a minimal promoter. The STAT3 reporter is premixed with constitutively expressing Renilla luciferase vector, which serves as an internal control for transfection efficiency._x000D_ The kit also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains the firefly luciferase gene under the control of a minimal promoter, without any additional response elements. The negative control is critical to determining pathway-specific effects and background luciferase activity.
Description: The SRE Reporter - HEK293 cell line is designed for monitoring the activity of the JAK/STAT signaling pathway. The SRE Reporter - HEK293 cell line contains a firefly luciferase gene under the control of SRE responsive elements stably integrated into HEK293 cells, resulting in an ERK pathway-responsive reporter cell line.
Description: The TEAD Reporter andndash; MCF7 cell line contains the firefly luciferase gene under the control of TEAD responsive elements stably integrated into the human breast cancer cell line, MCF7. Inside the cells, basal unphosphorylated YAP/TAZ remains in the nucleus and induces the constitutive expression of luciferase reporter. The cell line is validated for the inhibition of the expression of luciferase reporter by the activators of the Hippo pathway.
Description: The TCF/LEF Reporter kit is designed for monitoring the activity of Wnt / β-catenin signaling pathway in the cultured cells. The kit contains transfection-ready TCF/LEF luciferase reporter vector, which is a Wnt pathway-responsive reporter. This reporter contains a firefly luciferase gene under the control of multimerized TCF/LEF responsive element located upstream of a minimal promoter. The TCF/LEF reporter is premixed with constitutively-expressing Renilla luciferase vector that serves as internal control for transfection efficiency. _x000D_The kit also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, without any additional response elements. The negative control is critical to determining pathway specific effects and background luciferase activity. _x000D_
Description: The SRE Reporter Kit is designed for monitoring the activity of the ERK signaling pathway and the transcriptional activity of SRF in cultured cells. The kit contains a transfection-ready SRE luciferase reporter vector, which is an ERK pathway-responsive reporter. This reporter contains the firefly luciferase gene under the control of multimerized SRE responsive elements located upstream of a minimal promoter. The SRE reporter is premixed with a constitutively-expressing Renilla (sea pansy) luciferase vector that serves as an internal control for transfection efficiency. The kit also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains the firefly luciferase gene under the control of a minimal promoter, without any additional response elements. The negative control is critical for determining pathway-specific effects and the background luciferase activity.
Description: The ISRE Reporter kit is designed for monitoring the activity of Type I interferon-induced JAK/STAT signaling pathway in the cultured cells. The kit contains transfection-ready ISRE luciferase reporter vector, which is a JAK/STAT pathway-responsive reporter. This reporter contains a firefly luciferase gene under the control of multimerized ISRE responsive element located upstream of a minimal promoter. The ISRE reporter is premixed with constitutively-expressing Renilla (sea pansy) luciferase vector, which serves as an internal control for transfection efficiency. The kit also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as a negative control. The noninducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, without any additional response elements. The negative control is critical to determining pathway specific effects and background luciferase activity.
Description: The SBE Reporter kit is designed for monitoring the activity of TGFβ/SMAD signaling pathway in the cultured cells. The kit contains transfection-ready SBE luciferase reporter vector, which is a TGFβ pathway-responsive reporter. This reporter contains a firefly luciferase gene under the control of multimerized SBE responsive element located upstream of a minimal promoter. The SBE reporter is premixed with constitutively-expressing Renilla luciferase vector that serves as internal control for transfection efficiency. The kit also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as negative control. The noninducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, without any additional response elements. The negative control is critical to determining pathway-specific effects and background luciferase activity.
We named this technique dual-terminal amplification (DTA) analysis. Results: By utilizing the DTA analysis, each in situ fluorescence imaging analysis and ex situ fluorescence quantitative analysis of MUC1 have been achieved. In addition, the aptamer-containing DNA ternary complicated stays on cell floor solely through the analysis and leaves the cell after the analysis is full. The cells could be maintained in a non-interfering state for the remaining of the time.
So after the analysis, it’s discovered that there aren’t any impact on the physiological exercise of cells and the expression of goal protein even after two rounds of repeatable imaging and quantitative analysis. Conclusion: In abstract, we’ve efficiently constructed a technique for nondestructive analysis of membrane protein in living cells.
We consider that this technique offers a promising approach for the analysis of the important thing membrane proteins of cells and the versatile utilization of treasured cell samples.
