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兔单抗亲和力和灵敏度更高
相对于鼠单抗（解离常数Kd在10-9-10-10M水平），兔单抗的亲和力可提高100-1000倍（Kd在10-10-10-12M水平）。实际使用时，兔单抗的工作浓度更低，灵敏度更高，因此产生的背景更低，大大降低了假阳性的发生。用作免疫组化等用途时，有时甚至可免去抗原修复的步骤。
兔单抗能识别更多表位
很多蛋白在人类和啮齿动物中同源性很高，这些保守表位在小鼠体内免疫原性很弱，不容易产生高质量抗体。而兔做为宿主，可以更好地识别这些表位，产生针对更多表位的抗体。当然，兔也适合生产针对鼠类蛋白的抗体。
Purification of Antiserum or Ascites by Protein A/G Chromatography
1、Required Materials and Equipment
(1) Protein A or G agarose gel column (10 ml or 5 ml of packed beads; see guidelines below for choice of protein A or protein G)
(2) Ice-cold Tris-buffered saline (TBS). See recipe below
(3) 5% sodium azide solution
(4) Neutralization Buffer (NB). See recipe below
(5) Elution Buffers (pH 2.7 and pH 1.9). See recipes below
(6) Centrifuge tubes and microcentrifuge tubes
(7) Preparative centrifuge and microcentrifuge
(8) pH strips
(9) Microtiter plate reader with 600 nm filter
(10) Glass Columns
2、Guidelines for Choosing Protein A Agarose or Protein G Agarose
(1) Antibodies bind with different affinities to protein A and protein G conjugated agaroses. Use the chart below to choose the best affinity agarose for your antibody
Species and Type of Antibody
Agarose
Rabbit IgG Protein A or Protein G
Mouse IgG1 Protein G
Mouse IgG2 Protein A or Protein G
Mouse IgG3 Protein G
Sheep IgG Protein G but binds weakly
Rat IgG Protein G but binds weakly
Guinea Pig IgG Protein A
Dog IgG Protein A
Goat IgG Protein G
Pig IgG Protein A
(2) Buffer Preparation
To prepare 1 liter of TBS (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.05% sodium azide) add the following to 800ml of distilled water:
Ⅰ6.06 g of Tris base
Ⅱ8.77 g of NaCl
Ⅲ 10ml of 5% sodium azide stock solution (Do not add if the antibody will used in assays of cellular response or function.)
Ⅳ Mix well and adjust the pH to 7.4 with 5 N HCl. Bring the volume up to 1 L with distilled water and recheck the pH after chilling on ice.
(3) To prepare 100 ml of NB (1 M Tris-HCl, pH 8.0; 1.5 M NaCl; 1mM EDTA; 0.5% sodium azide), add the following to 80 ml of distilled water
Ⅰ12.1 g of Tris base
Ⅱ 8.7 g of NaCl
Ⅲ 200 microliters of 0.5M EDTA
Ⅳ 10 ml of 5% sodium azide stock solution (Do not add if the antibody will used in assays of cellular response or function.
Ⅴ Mix thoroughly and adjust the pH to 8.0 with 5 N HCl. Add distilled water to obtain a final volume of 100 ml. The NaCl and EDTA are added to prevent aggregation of the antibodies and loss of biological activity.
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