Axin antibody C8 preparation method
A technology of antibody and polyclonal antibody, applied in the direction of anti-animal/human immunoglobulin, recombinant DNA technology, using vectors to introduce foreign genetic material, etc., can solve the problem of low expression of endogenous Axin, and achieve unique and specific methods Strong, low-cost effect
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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 751-832. When designing PCR primers, Nco I and Xho I restriction sites were added at the 5' end and 3' end respectively. The primer sequences are as follows:
[0016] Primer F: 5'-ccgccatggagcattgttgtgggctac-3'
[0017] Primer R: 5'-ccgctcgaggtccaccttttccaccttg-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: cycle denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, cycle 30 time...
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. 8 μl of each sample was electrophoresed on a 10% SDS-PAGE gel. The same sample was run in two lanes respectively, and western blot analysis was performe...
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 Lyse the cells with 500 μl of lysate at 4°C for 5 minutes, sonicate the cells, centrifuge at 13,200 rpm at 4°C for 25 minutes, take 450 μl of supernatant, add 5 μl of anti-Axin antibody C8, follow the literature (Zhang Y et al., [J].J Biol Chem 1999 , 274: 35247-35254.) for immunoprecipitation, after washing the antigen-antibody-A / G agarose bead complex, add 30 μl cell lysate and 35 μl 2×SDS sample buffer, boil for 5 minutes, take 8 μl sample for SDS - PAGE gel electrophoresis, western blot analysis, wherein the primary antibody is anti-Axin antibody C8 diluted 1:1000, the results are shown in Figure 3. The left lane in the figure is the myc-Axin sample in Figure 2, the middle lane is t...
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