Anti-mouse Stk40 polyclonal antibody and preparation thereof
A polyclonal antibody, mouse technology, applied in the direction of peptide source, animal/human peptide, etc., can solve the problem that the antibody effect cannot meet the requirements of Western blotting application.
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[0059] More specifically, the preparation method of the present invention includes the steps of constructing an antigen expression vector containing the polypeptide, transforming, inducing expression, and purifying to obtain the antigen, immunizing, and purifying the antibody.
[0060] The antigens of the present invention can be prepared by methods well known in the art. For example, an expression vector for the antigen can be constructed, and then a host cell can be transformed with the expression vector so that the host cell can express the antigen, and the antigen can be isolated and purified from the host cell culture.
[0061] There is no special limitation on the choice of expression vector, as long as it can express the antigen of the present invention in host cells. Generally, an appropriate expression vector can be selected according to the polypeptide actually used to facilitate purification. For example, when using GST fusions, the PGEX-4T-1 vector can be used as ...
Embodiment 1
[0069] Example 1: Construction of antigen expression vector
[0070] The Stk40-C72 antigen encodes a total of 72 amino acids at positions 349-420 of the C-terminal of Stk40, and its sequence is shown in SEQ ID NO:4.
[0071] The construction process of Stk40-C72 antigen expression vector is as follows:
[0072] 1. Design cloning primers introducing restriction enzyme sites:
[0073] C72-F (BamH I): 5'TTG GGA TCC ATG CCG GAC ATT GAT GAC 3' (SEQ ID NO: 2)
[0074] C72-R (Xho I): 5' TGT CTC GAG ACT ACA TTG GCT GTG CGT 3' (SEQ ID NO: 3)
[0075] 2. Using the Flag-LYK4 / ppyCAGIP plasmid (Lingjie Li * 、Lei Sun * , et al., Stk40 Links the Pluripotency Factor Oct4 to the Erk / MAPK Pathway and Controls Extraembryonic Endoderm Differentiation PNAS.2010 Jan; 107(4):1402-1407) as a template, using C72-F (BamH I) and C72-R ( Xho I) primers for PCR to obtain a 210bp target fragment; [Note: LYK4 is Stk40]
[0076] 3. Digest the PCR product with BamH I and Xho I, and perform gel recovery....
Embodiment 2
[0080] Example 2: Antigen Purification
[0081] 1. Transform the LYK4-C72 / pGEX-4T-1 plasmid prepared in Example 1 into BL21 competent;
[0082] 2. Pick the bacteria into 5ml YTA (peptone 16g / L; yeast extract 10g / L; sodium chloride 5g / L) and shake overnight;
[0083] 3. Inoculate 2L YTA at 1:30 and shake for 3h; 0.2mM (GST) or 1mM (His) IPTG induction at 30°C for 3h;
[0084] 4. Collect bacteria by centrifugation at 10000g for 3 minutes, divide into 4 tubes, resuspend with 15ml PBS (GST) respectively, add PMSF (final concentration 1mM) at 1:100;
[0085] 5. Ultrasound, intensity: 10-12; time: 8-10min; interval: 2s;
[0086] 6. After ultrasonication, add 1% TritonX-100, centrifuge at 10000g for 10min, and take the supernatant;
[0087] 7. Handle GST beads during centrifugation: wash 3 times, 10 times the volume of PBS, 500g, 5min; (storage: 80%)
[0088] Mix the centrifuged bacterial supernatant with the treated GST beads (5-10 mg protein per ml), and rotate at 4°C for 2-4 h...
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