Method for preparing antibody C3 against Axin
An antibody and polyclonal antibody technology, applied in anti-animal/human immunoglobulin, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problem of low expression of endogenous Axin, and achieve unique methods and processes Simple, antibody-specific effects
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Embodiment 1
[0013] Example 1: Preparation of Antibodies
[0014] 1.PCR to obtain the target gene fragment
[0015] The template used was the Norwegian mouse Axin1 gene sequence (Genbank No. NM_024405), and the PCR fragment was a DNA fragment encoding the amino acid sequence of Axin348-432. When designing PCR primers, NcoI and Sa / I restriction sites were added at the 5' end and 3' end respectively. The primer sequences are as follows:
[0016] Primer F: 5'-aatccatggcccccatacaggatccg-3'
[0017] Primer R: 5'-ccggtcgacggcatttcaccatcctcc-3'
[0018] PCR amplification system (50μl):
[0019] Template pCMV-myc-Axin (0.01μM) 0.5μl
[0020] Primer F (0.2μM) 1μl
[0021] Primer R (0.2μM) 1μl
[0022] dNTP (2.5mM) 1μl
[0023] 10×Pyrobest buffer 5μl
[0024] ddw 41.2μl
[0025] Pyrobest TM DNA polymerase (purchased from Takara Company Dalian Branch) 0.3 μl
[0026] PCR reaction cycle: denature at 94°C for 30s, anneal at 60°C for 30s, extend at 72°C for 30s, and cycle 30 times.
[0027]...
Embodiment 2
[0066] HEK 293T cells (human embryonic kidney cells) were cultured in DMEM medium (purchased from Gibco, USA) (containing 10% calf serum) and grown in a 5% carbon dioxide incubator. Cells with an appropriate concentration were spread on two 60mm cell culture plates, and 2 μg of pCMV5 empty vector and wild-type Axin expression vector pCMV-myc-Axin were respectively transfected into 293T cells according to the conventional calcium phosphate precipitation method. After 36 hours, according to the literature ( Zhang Y et al., [J]. J Biol Chem 1999, 274: 35247-35254.) collected the cells, lysed the cells with 500 μl of cell lysate, and after a brief sonication, centrifuged the sample at 13200 rpm for 25 minutes at 4°C, and took the upper Clear 100 μl, add an equal volume of 2×SDS loading buffer, boil in boiling water for 5 minutes. Load 4 μl of each sample on a 10% SDS-PAGE gel for electrophoresis. The same sample was run in two swimming lanes respectively, and Western blot analysi...
Embodiment 3
[0068] Spread appropriate concentration of HEK 293T cells on five 60mm cell culture plates. After the cells are full, discard the cell culture medium, scrape off the cells with a cell scraper, wash with PBS three times, and collect the cells by centrifugation at 500g. Use the cells in Example 1 Cells were lysed with 500 μl of lysate at 4°C for 5 minutes. After the cells were disrupted by ultrasound, centrifuged at 13200 rpm at 4°C for 25 minutes, 450 μl of supernatant was taken, and 5 μl of anti-Axin antibody C3 was added. According to the literature (Zhang Y et al., [J].J Biol Chem 1999, 274: 35247-35254.) Perform immunoprecipitation, wash the antigen-antibody-A / G agarose bead complex, add 30 μl cell lysate and 35 μl 2×SDS sample buffer, boil for 5 minutes, and take 8 μl sample Carry out SDS-PAGE gel electrophoresis and western blot analysis, wherein the primary antibody is anti-Axin antibody C3 diluted 1:1000, the results are as follows image 3 . The left lane in the figur...
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