Small interfering RNA of fatty acid binding protein 4 and its application
A small interference and complex technology, applied in the field of biotechnology and medicine, can solve the problems of unseen intervention in the treatment and prevention of tuberculosis, and achieve the effect of promoting apoptosis
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[0083] Example 1: Changes in the expression of FABP4 after BCG infection of RAW264.7 cells
[0084]The cells were collected after 6h, 12h, 18h, and 24h of BCG infection (MOI 1:10) respectively, and the total protein of RAW264.7 cells was extracted. A negative control group (Negative Control, NC, wild RAW264.7 cells) was set in the experiment at the same time.
[0085] Specific steps are as follows:
[0086] a) Protein extraction: The protein extraction was carried out according to the operation steps of the whole protein extraction kit instructions (Keygen Biotechnology, Nanjing). The concentration of the extracted protein was detected by BCA kit (Thermo fisher, American), and 20 μg of sample was loaded into each well for subsequent Western-Blot detection;
[0087] b) Western-Blot detection: the separation gel concentration was selected as 12%. After electrophoresis, the proteins on the gel were electrotransferred to PVDF membrane (0.2 μm, BD company), and then blocked with 5...
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[0089] Example 2: Observation of the expression of FABP4 in each treatment group by immunofluorescence
[0090] The experimental group was divided into four groups, namely Control group (RAW264.7 cells), BCG group (RAW264.7 cells infected with BCG), siRNA-FABP4 group (RAW264.7 cells transfected with siRNA-(A+B)) and siRNA -FABP4+BCG group (RAW264.7 cells were transfected with siRNA-(A+B) and then infected with BCG), in which the Control group and BCG group were transfected with siRNA-NC, and the siRNA-FABP4 group and siRNA-FABP4+BCG group were transfected with siRNA -FABP4, infected with or without BCG after 24h, treated by immunofluorescence after 12h, and then observed by confocal microscope.
[0091] Specific steps are as follows:
[0092] Cell crawl:
[0093] a) The day before infection, 5 × 10 per well 4 Cells were seeded on cell slides in twelve-well plates, 1 mL of DEME medium containing 10% FBS was added to each well, and the cells were incubated at 37°C, saturated ...
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[0109] Example 3: Western-Blot detection of differences in the expression of FABP4 in each group
[0110] The setting of the experimental treatment group and the specific treatment time are the same as those in Example 2. After the treatment, the total protein of RAW264.7 cells in each treatment group was extracted, and the expression of FABP4 in the cells was detected by Western-Blot. Protein extraction, BCA quantification and Western-Blot specific The test operation was the same as that of Example 1. According to the steps described in the "Technical Method Description" section above, use the primary antibody (rabbit anti-rabbit FABP4 antibody, Thermo fisher, Prod#701158) to incubate overnight at 4°C, wash with the secondary antibody (goat anti-rabbit HRP enzyme-labeled secondary antibody, Proteintech) , SA00001-2) for Western-Blot detection. See the results image 3 , among which, the four columns from left to right are: Control group, BCG group, siRNA-FABP4 group and siR...
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