An rcan1 adapter rcan1-s14
A technology of rcan1-s14 and aptamer, which is applied in the field of biomedicine, can solve the problems of long-term enhancement effect damage, weakening of related signal memory, etc., and achieve the effect of weakening biological functions
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0047] Example 1 Intracellular localization of RCAN1
[0048] The RCAN1.1S full-length plasmid pRCAN1.1s-FL-GFP and the truncated plasmid pRCAN1.1s-1-103aa-GFP (plasmid map as shown in Figure 13 As shown, the sequence inserted into pRCAN1.1s-FL-GFP is SEQ ID NO: 2, and the sequence inserted into pRCAN1.1s-1-103aa-GFP is SEQ ID NO: 3) transfected into HEK293 cells (human kidney epithelial cells) , Collect the cells after 48h. The nuclei and cytoplasm were separated by the nuclear protein and cytoplasmic protein extraction kit (P0027, Beyond), and the distribution of the two in the cells was detected by Western blotting using anti-GFP antibody (1:000, G1546, Sigma). The result is as figure 1 with figure 2 As shown, it can be seen from the figure that the distribution of RCAN1.1S-FL and RCAN1.1s-1-103aa in the nucleus is greater than that in the cytoplasm.
[0049] pRCAN1.1s-FL-GFP insertion sequence:
[0050] ATGGAGGAGGTGGACCTGCAGGACCTGCCCAGCGCCACCATCGCCTGTCACCTGGACCCGCGCGT...
Embodiment 2
[0055] Example 2 RNaseA Sensitivity of RCAN1
[0056] We transfected HEK293 cells with RCAN1.1S-FL-GFP and RCAN1.1S1-103aa-GFP respectively, and added RNaseA (1mg / ml, 000000010109169001, Roche) after 24 hours of transfection for 7 minutes at 37°C, and then collected and Cells were lysed, and we detected the expression levels of the two using anti-GFP antibody (1:000, G1546, Sigma) by Western blot technique, and the results were as follows image 3 shown. At the same time, we also used a confocal microscope (model LSM780) after immunofluorescent staining to observe whether the contents of the two changed. The results are as follows: Figure 4 shown. We planted the cells on glass slides, ruptured the membrane 24 hours after transfection, washed twice with PBS, added RNaseA (1mg / ml) at 37°C for 7 minutes, fixed with cold formaldehyde for 15 minutes, and then used DAPI (4',6-diamidino Base-2-phenylindole) to stain the nuclei, and finally use confocal technology to observe the e...
Embodiment 3
[0057] Example 3 Purification of RCAN1.1S-1-103aa protein
[0058] We transformed the constructed prokaryotic expression vector pet-RCAN1-1-103 into BL21(DE3) competent cells, picked a single colony and amplified the bacteria to 10ml of LB with antibiotics added and shook overnight at 37°C. Dilute the overnight bacteria at a ratio of 1:100, that is, add 10ml of the overnight bacteria to 1L of LB and incubate at 37°C for 2-3h, then add 0.5mM IPTG (ST098, Biyuntian) to induce protein expression. After culturing overnight at 37°C, the bacteria were collected, lysed and centrifuged, and the protein was separated and purified through a Ni-NTA Resin affinity chromatography column to obtain RCAN1.1S-1-103 protein. The purified protein was detected by Western blot method, and the detection results were as follows: Figure 5 As mentioned above, it can be seen from the figure that the prokaryotic expression vector pet-RCAN1-1-103 constructed by us can successfully express the RCAN1.1S-1...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More