產品名稱：Protein A agarose Resin 6FF for Antibody Purification， Protein A抗體純化填料

貨號： P2108

品牌：Engibody

簡介：Protein A Resin, Protein A抗體純化填料用于人和兔的單克隆及多克隆抗體的純化。

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偶聯瓊脂糖6FF是一種用于抗體純化的多功能、高性能親和樹脂，可用作瓶裝瓊脂糖珠、預包裝柱和完整的IgG純化試劑盒。

蛋白A瓊脂糖6FF由純化的重組蛋白A組成，重組蛋白A以高密度共價固定在高質量交聯的6%珠狀瓊脂糖上。在許多可用的固定化蛋白A親和樹脂品種中，這一種為從哺乳動物血清樣品中高產量和高純度純化整個IgG提供了通用的色譜特征組合。瓊脂糖珠具有適用于許多親和純化系統的物理和化學性質。因此，Protein A Sepharose 6FF以幾種方便的包裝形式提供，包括瓶裝樹脂漿、三種尺寸的旋轉柱、完整的純化試劑盒、兩種尺寸的FPLC色譜柱。

蛋白A是一個46.7kDa的Staphylococcus aureus細菌細胞壁蛋白，由一條基本上不含碳水化合物的多肽鏈組成。天然蛋白A含有四個高親和力（Ka=10^8/mol）結合位點，能夠與來自選定哺乳動物物種的IgG類抗體的Fc區相互作用。IgG結合功能在pH 8.2時最佳，但在中性和生理緩沖液（pH 7.0至7.6）中也發生有效結合。

與替代品（蛋白G）相比，蛋白A為兔、豬、狗和貓的IgG亞類提供了最高的結合能力。蛋白A對人IgG的結合能力也更高，但IgG3的結合能力較弱。蛋白A與小鼠IgG1弱結合，不推薦用于該抗體同種型的大多數應用。

經過測試的應用實驗

抗體純化

類型

50%懸液

存儲說明

2-8°C，請勿結冰

beads直徑

45微米-165微米

結合能力

>40 mg人IgG/mL沉淀樹脂

配體

大腸桿菌中產生的重組蛋白A

基質

高交聯6%瓊脂糖珠子

最大壓力

0.3 MPa（3bar）

化學穩定性

所有常用的水性緩沖液，包括6M鹽酸胍和8M尿素。

流速

重力流，100-300 cm/小時

pH穩定性

長期：pH3-9；

短期：pH2-10

存儲緩沖液

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.

抗體純化方案（重力流柱）

注：以下方案適用于使用填充有1mL蛋白A瓊脂糖珠（即2mL 50%珠漿）的重力流柱。當使用含有其他樹脂體積的柱時，必須相應調整試劑量。

所需的其他試劑和材料

•床體積為10-15 mL的重力流柱

•15mL收集管

•結合緩沖液：100mM磷酸鹽，150mM氯化鈉；溶于500mL超純水時pH 7.2

•IgG洗脫緩沖液：0.1M甘氨酸，pH 2-3

•中和緩沖液：12mL，1M Tris•HCl，pH 8.5

•儲存溶液：0.02%抑菌劑磷酸鹽緩沖鹽水（PBS，pH 7.4）

抗體純化步驟

1.將重力流空柱、蛋白A瓊脂糖珠和緩沖液平衡至室溫。

2.在使用蛋白A瓊脂糖珠柱純化之前，用結合緩沖液將樣品稀釋至少1:1，以保持適當的離子強度和pH以實現最佳結合。

注：由于脂蛋白沉淀，血漿在用結合緩沖液稀釋后可能變得模糊。以10000×g離心稀釋樣品20分鐘，并將上清液涂在平衡的蛋白A瓊脂糖珠上。

3.用2mL樹脂漿料輕輕填充柱子，并讓儲存溶液排出。

4.通過添加5mL結合緩沖液平衡柱，并使溶液通過柱排出。

5.將稀釋后的樣品涂在色譜柱上。為了獲得最佳結果，添加小于柱抗體結合能力80%的樣品體積。收集流量。

注：如果樣品中的IgG含量超過了可以與柱結合的水平，則流通液中將含有過量的抗體。通過保存流通，可以回收和分析未結合的抗體。

6.用15mL結合緩沖液洗滌柱。

注：如果需要，通過收集分離的2mL級分作為柱排出物，驗證是否從柱中去除了所有未結合的蛋白質。在280nm處測量各組分的吸光度。最后一部分的吸光度應類似于結合緩沖液。

7.向收集管中加入100μL中和緩沖液。用5mL洗脫緩沖液洗脫抗體，在含有收集管的中和緩沖液中收集0.5-1mL級分。通過在280nm處測量吸光度或通過蛋白質測定（如BCA蛋白質測定）監測洗脫。

8.匯集含有最高吸光度的洗脫IgG級分。純化的抗體可以直接用于SDS-PAGE，或者可以通過透析或脫鹽柱將緩沖液交換成與特定下游應用兼容的緩沖液。

9.通過加入15mL洗脫緩沖液使柱再生，并使溶液流過柱。柱可以再生至少10次而不會顯著喪失結合能力。

10.通過添加5ml儲存溶液儲存柱。當柱中剩余約3mL時，蓋上底部并固定頂蓋。將色譜柱儲存在4°C。