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 3^rd 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