Axin antibody C8 preparation method
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 and high Specific, easy-to-use effects
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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, NcoI and XhoI 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 times....
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 swimming lanes respectively, and Western blot analysis was...
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 the supernatant was taken, and 5 μl of anti-Axin antibody C8 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 2xSDS sample buffer, boil for 5 minutes, take 8 μl sample for SDS -PAGE gel electrophoresis, westernblot analysis, wherein the primary antibody is anti-Axin antibody C8 diluted 1:1000, the results are as follows image 3 . The left lane in the figure is fi...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More