[박상규] Antibody production from Rabbit

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      Anonymous
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      Antibody production from Rabbit

                                                                   2005.3.03        Arthur

      1.     Purchase NewZealand White rabbit (male, 1kg)

      2.     Adapt rabbits for 3-7days

      3.     Pick up the blood (200-300ul) from ear vein using 1ml syringe and store at 4 degree or on ice for 1h

      4.     Centrifuge the blood at 14,000 rpm for 5 min

      5.     Take the supernatant and transfer the sup to new tube

      6.     Store at 70 degree till use.

       

      Blood test

      1.     Run the 10% SDS-PAGE with human cell line whole cell lysate (40ug/laneXtest serum)

      2.     Transfer the PVDF membrane

      3.     Block the membrane with 5% milk, 0.5% Tween 20 in TBS for 1 h at room temperature

      4.     Dilute the serum (1/1000) with 1% milk, 0.2% Tween 20 in TBS and bind the serum solution for 1h at RT

      5.     Wash the membrane with 1% milk, 0.2% Tween 20 in TBS for 10min, 2 times

      6.     Bind the 2ndary antibody solution(dilute the anti-rabbit-HRP with 1/25,000 to 1% milk, 0.2% Tween 20 in TBS) for 1 h at RT.

      7.     Wash the membrane with 1% milk, 0.2% Tween 20 in TBS for 10min, 2 times

      8.     Develop the membrane

       

      è Choose the non-reactive serum

      è Go for injection

       

      Injection and harvest step

      1.     Mix the protein (500ug protein(1mg/ml) to 0.5ml complete adjuvant) using syringe linker

      2.     Inject the mixed solution into 2 or 3 areas of sub-cutaneous of rabbit dorsum (300-400ul per area)

      3.     1 week later, Mix the protein (500ug protein(1mg/ml) to 0.5ml incomplete adjuvant) using syringe linker

      4.     Inject the mixed solution into 2 or 3 areas of sub-cutaneous of rabbit dorsum (300-400ul per area)

      5.     Repeat step 3, 4

      6.     3 days later after 3rd injection, pick up the blood (200-300ul) from ear vein to check if the antibody is generated

      7.     Run the antigen (50ng/lane) and human cell line whole cell lysate(40ug/lane) to check if the generated antibody is antigen specific and could recognize the right molecular weight in whole cell lysate. Basically, follow the western procedure. Dilute the serum with 1/3000 and 1/6000. If the antibody is antigen specific and could recognize the right molecular weight, go for step 9

      8.     You should perform the step 6, 7 within 1 day.

      9.     4 days later, finally boost the antibody following the step 3, 4.

      10.  3 days later, pick up the blood through the cardiac puncture using 50ml syringe and 18 guage needleè you can get the blood about 70ml-150ml. Store the blood on ice or 4 degree for overnight.

      11.  Centrifuge the blood at 26,000xg for 30 min at 4 dgree

      12.  Harvest the serum and make an aliquote (5ml and 1ml)

      13.  Store at 80 degree

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