Preparation method and application of antibody of membrane protein CD14
A membrane protein and antibody technology, which is applied in the fields of peptide preparation methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem that antibodies cannot recognize membrane proteins expressed by cells well
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Embodiment 1
[0024] A method for preparing an antibody to the membrane protein CD14 of this embodiment comprises the following steps:
[0025] Step (1): Obtain the CD14 antibody heavy chain and light chain variable region sequences:
[0026]The Balb / c mice were immunized three times with human peripheral blood mononuclear cells (PBMC) as the immunogen, and a booster immunization was performed 3 days before the spleen of the mice was taken to measure the titer of antiserum. Extract mRNA from tissues, reverse transcribe to obtain antibody cDNA, design specific primers according to the conserved sequences at both ends of the antibody variable region, refer to the nucleotide sequence list for primer sequences, and amplify the heavy chain variable region with primer VH pair and primer VL pair respectively and light chain variable region, the amplification reaction conditions are as follows: pre-denaturation at 94°C for 5 min, 94°C for 45 s, 58°C for 1 min, 72°C for 45 s, a total of 30 cycles, a...
Embodiment 2
[0037] A method for purifying the recombinant CD14 antibody prepared in Example 1, comprising the steps of:
[0038] 1) Take the affinity chromatography column, first wash the column bed with water, and then wash the chromatography column with sodium acetate buffer;
[0039] 2) Take the cell culture medium, centrifuge at 4°C and 12000rpm for 10min, collect the supernatant, and filter it with a 0.45μm filter membrane; 3) Take 5mL of the cell culture medium supernatant and mix it with an equal amount of sodium acetate buffer, and dilute with 0.5 Load the sample at the speed of mL / min, and collect the breakthrough; 4) After the sample loading, continue to wash with sodium acetate buffer until G250 detects colorless, wash the column bed with glacial acetic acid elution buffer, collect the elution peak, and quickly wash with saturated carbonic acid Sodium adjusts the elution peak pH to neutral; 5)
[0040] Wash the chromatographic column with 10 times the volume of ultrapure water...
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