Construction method, expression method and purification method of eukaryotic expression vector of human PAK1 protein
A technology for eukaryotic expression vectors and construction methods, which is applied in the field of expression and purification, and the construction of eukaryotic expression vectors. It can solve the problems of poor antibody specificity, low sensitivity, and poor specificity in recognizing natural PAK1 proteins, and achieve high specificity and sensitivity. High, high protein purity effect
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[0038] Example 1 Pak1 protein eukaryotic expression vector and its expression, purification
[0039] 1. Construction of PAK1 protein eukaryotic expression vector
[0040] The primer is designed according to the sequence of PAK1 proteins:
[0041] Upstream primers PAK1-F (SEQ ID NO.1): 5'-agctcaactatgatctttt-3 ',
[0042] Downstream primers PAK1-R (SEQ ID No.2): 5'-gaAttttcatctcctttt-3 '.
[0043] Among them, the BamHi enzyme digestion is introduced, and the EcoRi enzyme digestion and 6 × histidine label are introduced in the downstream primer.
[0044] HeLa cells were used as a template, and PCR amplification was performed on PAK1-F / PAK1-R using primers, and the reaction system was: PCR reaction MIX 25 μL, the concentration of 10 μm is 1 μL, DNA template 2 μL, DDH 2 O-50 μL. The PCR amplification procedure is: 95 ° C 180 seconds; 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, 35 cycles; last 72 ° C 300 seconds.
[0045] The PCR amplification was carried out...
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[0060] Example 2
[0061] First, the molecular cloning of PAK1 gene
[0062] The PCR amplification of PAK1-F / PAK1-R was performed using HeLa cells, and PCR amplification was performed on the primers in Example 1, and the reaction system was: PCR reaction MIX 25 μL, and the concentration of 10 μm was 1 μl, DNA template 2 μL, Plus DDH 2 O-50 μL. The PCR amplification procedure is: 95 ° C 180 seconds; 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, 35 cycles; last 72 ° C 300 seconds.
[0063] The PCR amplification was carried out by agarose gel electrophoresis, and the gel recovery kit was used to purify the PCR amplification product according to the PGEM-TEASY vector product specification. The reaction buffer was established in accordance with the PGEM-TEASY vector system product specification: 10 × T4 DNA connection reaction buffer 1 μL, PGEM-T vector 1 μL (50 ng), purified PCR amplification fragment 3 μL, T4 DNA ligase 1 μL (3U), DDH 2 O to a total volume of...
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