产品名称：Protein A agarose Resin 6FF for Antibody Purification， Protein A抗体纯化填料

货号： P2108

品牌：Engibody

简介：Protein A Resin, Protein A抗体纯化填料用于人和兔的单克隆及多克隆抗体的纯化。

Protein A Sepharose 6FF is a versatile, high-performance affinity resin for antibody purification that is available as bottled sepharose beads, pre-packed columns, and complete IgG purification kits.

Protein A Sepharose 6FF consists of purified recombinant Protein A that has been covalently immobilized at high density onto high-quality crosslinked 6% beaded sepharose. Among the many available varieties of immobilized Protein A affinity resins, this one provides the most versatile combination of chromatographic features for high yield and high purity purification of whole IgG from mammalian serum samples. The sepharose beads have physical and chemical properties suitable for many affinity purification systems. Accordingly, Protein A Sepharose 6FF is offered in several convenient package formats, including bottled resin slurries, three sizes of spin columns, complete purification kits, two sizes of FPLC-ready chromatography cartridges.

Protein A is a cell wall component produced by several strains of Staphylococcus aureus. The 46.7kDa-protein consists of a single polypeptide chain that is essentially devoid of carbohydrate. Native Protein A contains four high-affinity (Ka = 10^8/mol) binding sites that are capable of interacting with the Fc region of IgG-class antibodies from selected mammalian species. IgG-binding function is optimal at pH 8.2, but efficient binding also occurs in neutral and physiological buffers (pH 7.0 to 7.6).

Compared to its alternative (Protein G), Protein A provides the highest binding capacity for subclasses of IgG from rabbits, pigs, dogs and cats. Protein A also has higher binding capacity for human IgG, except for IgG3, which it binds weakly. Protein A binds weakly to mouse IgG1 and is not recommended for most applications with that antibody isotype.

Tested applications

Antibody Purification

Form

50% Slurry

Storage instruction

2 - 8°C, do not freeze

Beads Diameter

45 μm - 165 μm

Binding Capacity

>40 mg human IgG/mL settled resin

Ligand

Recombinant protein A produced in E.coli

Matrix

Highly cross-linked 6% agarose beads

Maximum pressure

0.3 MPa (3 bar)

Chemical stability

All commonly used aqueous buffers, including 6 M guanidine-HCl and 8 M urea.

Flow rate

Gravity flow, 100 - 300 cm/hour

pH stability

Long term: pH3 - 9;

Short term: pH2 - 10

Storage buffer

1x PBS containing 20% ethanol

蛋白A偶联琼脂糖6FF是一种用于抗体纯化的多功能、高性能亲和树脂，可用作瓶装琼脂糖珠、预包装柱和完整的IgG纯化试剂盒。

蛋白A琼脂糖6FF由纯化的重组蛋白A组成，重组蛋白A以高密度共价固定在高质量交联的6%珠状琼脂糖上。在许多可用的固定化蛋白A亲和树脂品种中，这一种为从哺乳动物血清样品中高产量和高纯度纯化整个IgG提供了通用的色谱特征组合。琼脂糖珠具有适用于许多亲和纯化系统的物理和化学性质。因此，Protein A Sepharose 6FF以几种方便的包装形式提供，包括瓶装树脂浆、三种尺寸的旋转柱、完整的纯化试剂盒、两种尺寸的FPLC色谱柱。

蛋白A是一个46.7kDa的Staphylococcus aureus细菌细胞壁蛋白，由一条基本上不含碳水化合物的多肽链组成。天然蛋白A含有四个高亲和力（Ka=10^8/mol）结合位点，能够与来自选定哺乳动物物种的IgG类抗体的Fc区相互作用。IgG结合功能在pH 8.2时最佳，但在中性和生理缓冲液（pH 7.0至7.6）中也发生有效结合。

与替代品（蛋白G）相比，蛋白A为兔、猪、狗和猫的IgG亚类提供了最高的结合能力。蛋白A对人IgG的结合能力也更高，但IgG3的结合能力较弱。蛋白A与小鼠IgG1弱结合，不推荐用于该抗体同种型的大多数应用。

经过测试的应用实验

抗体纯化

类型

50%悬液

存储说明

2-8°C，请勿结冰

beads直径

45微米-165微米

结合能力

>40 mg人IgG/mL沉淀树脂

配体

大肠杆菌中产生的重组蛋白A

基质

高交联6%琼脂糖珠子

最大压力

0.3 MPa（3bar）

化学稳定性

所有常用的水性缓冲液，包括6M盐酸胍和8M尿素。

流速

重力流，100-300 cm/小时

pH稳定性

长期：pH3-9；

短期：pH2-10

存储缓冲液

1x PBS，含20%乙醇

Antibody Purification Protocol (Gravity-flow column)

Note: The following protocol is for using a gravity-flow column packed with 1mL of Protein A agarose beads (i.e., 2mL of the 50% beads slurry). When using columns containing other resin volumes, reagent amounts must be adjusted accordingly.

Additional Solutions and Materials Required

• Gravity-flow column of 10-15 mL bed volume

• 15mL collection tubes

• Binding Buffer: 100mM phosphate, 150mM sodium chloride; pH 7.2 when dissolved in 500mL of ultrapure water

• IgG Elution Buffer: 0.1M glycine, pH 2-3

• Neutralization Buffer: 12mL, 1M Tris•HCl, pH 8.5

• Storage solution: 0.02% sodium azide in phosphate-buffered saline (PBS, pH 7.4)

Antibody Purification Procedure

1. Equilibrate column, protein A agarose beads and buffers to room temperature.

2. Dilute sample at least 1:1 with Binding Buffer before purification using the Protein A beads Column to maintain the proper ionic strength and pH for optimal binding.

Note: Plasma may become hazy upon dilution with the Binding Buffer because of lipoprotein precipitation. Centrifuge the diluted sample at 10,000 × g for 20 minutes and apply the supernatant to the equilibrated Protein A agarose beads.

3. Gently pack the column with 2mL of resin slurry and allow storage solution to drain.

4. Equilibrate column by adding 5mL of Binding Buffer and allow the solution to drain through the column.

5. Apply the diluted sample to the column. For best results, add a sample volume that is less than 80% of the column’s antibody-binding capacity. Collect the flow-through.

Note: If the sample contains more IgG than can bind to the column, the flow-through will contain the excess antibody. By saving the flow-through, nonbound antibody can be recovered and analyzed.

6. Wash column with 15mL of Binding Buffer.

Note: If desired, verify that all nonbound proteins are removed from the column by collecting separate 2mL fractions as the column drains. Measure the absorbance of each fraction at 280nm. The last fractions should have an absorbance similar to the Binding Buffer.

7. Add 100μL Neutralization Buffer to collection tubes. Elute antibodies with 5mL of Elution Buffer, collecting 0.5-1mL fractions in the neutralization buffer-containing collection tubes. Monitor the elution by measuring the absorbance at 280nm or by protein assay such as BCA Protein Assay.

8. Pool the eluted IgG fractions that contain the highest absorbance. The purified antibodies may be used directly for SDS-PAGE, or the buffer may be exchanged by dialysis or de salting column to one that is compatible with the specific downstream application.

9. Regenerate column by adding 15mL of Elution Buffer and allow solution to flow through the column. Columns may be regenerated at least 10 times without significant loss of binding capacity.

10. Store column by adding 5 ml of storage solution. When approximately 3mL remain in the column, cap bottom and secure top cap. Store column at 4°C.

抗体纯化方案（重力流柱）

注：以下方案适用于使用填充有1mL蛋白A琼脂糖珠（即2mL 50%珠浆）的重力流柱。当使用含有其他树脂体积的柱时，必须相应调整试剂量。

所需的其他试剂和材料

•床体积为10-15 mL的重力流柱

•15mL收集管

•结合缓冲液：100mM磷酸盐，150mM氯化钠；溶于500mL超纯水时pH 7.2

•IgG洗脱缓冲液：0.1M甘氨酸，pH 2-3

•中和缓冲液：12mL，1M Tris•HCl，pH 8.5

•储存溶液：0.02%抑菌剂磷酸盐缓冲盐水（PBS，pH 7.4）

抗体纯化步骤

1.将重力流空柱、蛋白A琼脂糖珠和缓冲液平衡至室温。

2.在使用蛋白A琼脂糖珠柱纯化之前，用结合缓冲液将样品稀释至少1:1，以保持适当的离子强度和pH以实现最佳结合。

注：由于脂蛋白沉淀，血浆在用结合缓冲液稀释后可能变得模糊。以10000×g离心稀释样品20分钟，并将上清液涂在平衡的蛋白A琼脂糖珠上。

3.用2mL树脂浆料轻轻填充柱子，并让储存溶液排出。

4.通过添加5mL结合缓冲液平衡柱，并使溶液通过柱排出。

5.将稀释后的样品涂在色谱柱上。为了获得最佳结果，添加小于柱抗体结合能力80%的样品体积。收集流量。

注：如果样品中的IgG含量超过了可以与柱结合的水平，则流通液中将含有过量的抗体。通过保存流通，可以回收和分析未结合的抗体。

6.用15mL结合缓冲液洗涤柱。

注：如果需要，通过收集分离的2mL级分作为柱排出物，验证是否从柱中去除了所有未结合的蛋白质。在280nm处测量各组分的吸光度。最后一部分的吸光度应类似于结合缓冲液。

7.向收集管中加入100μL中和缓冲液。用5mL洗脱缓冲液洗脱抗体，在含有收集管的中和缓冲液中收集0.5-1mL级分。通过在280nm处测量吸光度或通过蛋白质测定（如BCA蛋白质测定）监测洗脱。

8.汇集含有最高吸光度的洗脱IgG级分。纯化的抗体可以直接用于SDS-PAGE，或者可以通过透析或脱盐柱将缓冲液交换成与特定下游应用兼容的缓冲液。

9.通过加入15mL洗脱缓冲液使柱再生，并使溶液流过柱。柱可以再生至少10次而不会显著丧失结合能力。

10.通过添加5ml储存溶液储存柱。当柱中剩余约3mL时，盖上底部并固定顶盖。将色谱柱储存在4°C。