Preparation and application of enrichment column for immunoaffinity purification of recombinant protein tagged with flag peptide
A recombinant protein, enrichment column technology, applied in chemical instruments and methods, separation methods, solid adsorbent liquid separation, etc., can solve the problems of severe elution conditions, poor purification effect, metal ion shedding, etc. The effect of high density, high coupling rate and strong recognition ability
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Embodiment 1
[0044] Example 1 Preparation of Immunoaffinity Purified D-tag-specific Polyclonal Antibody
[0045] 1.1 Design of D-tag short peptide hapten
[0046] Using a short peptide composed of 8 amino acid residues, namely aspartyl-tyrosyl-lysyl-aspartyl-aspartyl-aspartyl-aspartyl-lysine, using These 8 amino acid residues are divided into two kinds and the cysteine sulfhydryl SH group is introduced in opposite directions to prepare sulfhydrylated D-tag short peptides.
[0047] 1.2 Preparation of D-tag short peptide immunogen
[0048] Take 5 mg / mL KLH 10 mL, add 100 μL saturated Na 2 CO 3 , then add 1 mL of 10% SDS, mix well, boil for 5 minutes to denature, then react with iodoacetyl-NHS to generate iodoacetylated KLH, remove excess iodoacetic acid by G25 Sephadex, add 55 mg of D-tag short peptide half For the antigen, adjust the pH to 8.5, react at room temperature for 60 minutes, and obtain the D-tag-KLH covalent bond complex, which is the immune antigen after desalting with G25...
Embodiment 2
[0061] Example 2 Preparation of enrichment column for immunoaffinity purification of recombinant protein with high-density D-tag tag.
[0062] 2.1 Transfer 3 mL of Agarose activated by sodium iodate to the column, wash with 100 mL of distilled water, drip dry, and pass through 0.1 mol / L Na 2 CO 3 , drip dry and set aside;
[0063] 2.2 Dilute 300 mg of immunoaffinity-purified D-tag-specific polyclonal antibody with 0.1 mol / L Na 2 CO 3 Dissolve to make the concentration reach 20 mg / mL, centrifuge to get the supernatant after complete dissolution;
[0064] 2.3 Add the supernatant to the affinity column in step 2.1, collect the effluent and pass through the column again, repeat 3-5 times;
[0065] 2.4 Wash out the unbound antibodies with 20 mL of distilled water and collect them together;
[0066] 2.5 Prepare 1.2 mL of sodium borohydride solution with a concentration of 10 mg / mL with distilled water, add it to the column, shake and mix, and react at 4°C for 30 minutes;
[00...
Embodiment 3
[0070] Example 3 D-tag-specific antibody-labeled horseradish peroxidase
[0071] 3.1 Weigh 5 mg immunoaffinity purified D-tag specific polyclonal antibody, dissolve in 100 μL 0.1 mol / L Na 2 CO 3 , react for 10 minutes, according to the amount of antibody: horseradish peroxidase = 1:1, add 5 mg of activated horseradish peroxidase, mix well;
[0072] 3.2 Add 1 mg of sodium borohydride, mix well and exhaust, place at -20°C for 1 minute, then move to 4°C for 2 hours;
[0073] 3.3 Desalted by G25 Sephadex desalting column;
[0074] 3.4 Add BSA so that the final concentration of BSA is 1 mg / mL, add 50% glycerol, and detect the titer of the labeled antibody by ELISA.
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