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Protein A是一种分子量约为42 kDa的蛋白质，由单一多肽链构成，不含二硫键。其结构包含多个高度同源的IgG结合结构域（通常为4-5个），每个结构域由约55-62个氨基酸残基组成。该蛋白具有显著的稳定性，能够耐受尿素、酸性条件（pH 2.5）甚至加热煮沸而不失活。其核心功能是与免疫球蛋白G的Fc段结合，这种结合具有高度特异性和亲和力。结合范围涵盖多种哺乳动物（如人、猪、兔、小鼠等）的IgG，但对不同物种和IgG亚型的结合能力存在差异。以人源与鼠源 IgG 为例，人 IgG1、IgG2、IgG4 亚型可与 Protein A 形成稳定结合复合物，而 IgG3 亚型因 Fc 片段关键位点突变导致结合能力缺失；鼠源 IgG1 亚型与 Protein A 的结合亲和力显著低于 IgG2a、IgG2b 等亚型。尽管存在上述结合差异性，Protein A 凭借其对多数常用模式生物 IgG 的高特异性结合能力，仍是抗体亲和纯化领域应用最广泛的配体之一。
Protein A 抗体纯化试剂盒是针对抗体常规纯化设计的标准化实验工具，可实现高效、可重复的免疫球蛋白（Immunoglobulin G, IgG）分离。本试剂盒通过将高纯度蛋白 A 偶联到高交联度聚苯乙烯 - 二乙烯基苯（Polystyrene-Divinylbenzene, PS-DVB）微球上发挥作用，可用于从杂交瘤细胞上清液中纯化 IgG 组分。
产品组分
| P1455932 | Components | 2mL | 10mL | 储存温度 | Quantity Per Test |
| P1455932A | 结合缓冲液(10×) | 50mL | 250mL | 2-8℃ | 5mL |
| P1455932B | 洗脱缓冲液(5×) | 10mL | 50mL | 2-8℃ | 1mL |
| P1455932C | 中和缓冲液 | 10mL | 50mL | 2-8℃ | 1mL |
| P1455932D | 蛋白A填料 | 2mL | 10mL | 2-8℃ | 2mL |
| P1455932E | 重力柱 | 1个 | 5个 | 2-8℃ | 1个 |
| P1455932F | 考马斯亮蓝染色液 | 10mL | 50mL | 2-8℃ | 1mL |
| P1455932G | 再生缓冲液 | 10mL | 50mL | 2-8℃ | 10mL (Optional) |
注：每个Test推荐Protein A resin使用量为2mL
使用方法
使用前，需用去离子水将各组分缓冲液稀释至 1× 浓度。具体操作可根据不同纯化工具的要求调整。
一．样品制备
在纯化之前，必须通过离心或 0.45 μm 过滤器过滤来澄清杂交瘤组织上清、血清或腹水样品，以免堵塞纯化柱，降低纯化柱寿命。
注：本试剂盒中Protein A填料的动态结合载量≥30 mg hIgG/mL，实际操作时建议IgG上样量不超过该动态载量的80%（即单根色谱柱单次上样IgG总量应≤24 mg），以避免填料过载并保证纯化效果。由于杂交瘤细胞克隆的表达特性不同，其组织培养上清中的抗体浓度可能存在较大波动，因此每次纯化的实际得量通常介于2-10 mg之间，具体数值需根据实验条件（如上清液抗体浓度、样品体积等）实际测定。
二．样品纯化
2.1 装柱
1. 将纯化柱垂直固定，确认下方垫片已水平固定。
2. 取 2mL Protein A resin加入柱管，待填料在柱底部堆积后，小心放入上层垫片并压实填料。
3. 取下下方堵头，使柱管内液体自然滴落，最终 Protein A 纯化柱的有效体积为 1mL。
注：Protein A 树脂中含 50% 保护液，填装前需充分混匀，防止影响纯化效率。
放置垫片时，需尽量保持柱内液面平整，避免填料与垫片间产生气泡。气泡会降低分离效率，并增加维持稳定流速所需的压力
2.2 平衡
取 1× 结合缓冲液（Binding buffer）倒满纯化柱（约 15 个柱体积），用于平衡柱体，
2.2 上样
取下纯化柱上盖，将澄清后的样品加入 Protein A 纯化柱中，使液体自然滴落。
注：若样品中抗体含量较低或样品体积较大，可通过增加上样次数、延长上样时间，提升抗体与填料的结合效率。
2.3 洗涤
取 1× 结合缓冲液（Binding buffer）倒满纯化柱（约 15 个柱体积），清洗柱内未结合的杂质，使液体自然滴落。
2.4 洗脱
1. 准备数个 1.5mL 离心管，向每个离心管中预先加入 10μL 中和缓冲液（Neutralization Buffer）。
2. 向每个纯化柱中加入 3mL 1× 洗脱缓冲液（Elution buffer，约 3 个柱体积），通过重力作用收集洗脱液。每管收集 8 滴洗脱液（约 0.5mL），依次操作直至洗脱完成。
2.5 透析:
1. 从每管洗脱液中取 2.5μL，与 10μL 考马斯亮蓝染色液（Coomassie Brilliant Blue Staining Solution）混合，观察抗体洗脱的梯度变化。
2. 收集抗体浓度较高的洗脱管，将其置于适宜的缓冲液中进行透析置换，最终获得高纯度的纯化 IgG。
三．纯化柱储存
3.1 抗体洗脱完成后，继续向柱内加入 2mL 1× 洗脱缓冲液（Elution buffer）洗涤，避免柱内残留抗体。
3.2 取 1× 结合缓冲液（Binding buffer）倒满纯化柱（约 15 个柱体积），平衡柱体，使液体自然滴落。
3.3 取 20% 乙醇倒满纯化柱（约 15 个柱体积），进行防腐处理，使液体自然滴落。
3.4 向柱内加入 5mL 20% 乙醇，盖上上盖并堵紧下方堵头，于 2-8℃条件下储存，下次纯化时可直接取出使用。
四．柱再生 （可选）
当纯化柱的性能出现可观测的下降时，即应执行再生程序，具体操作如下：
4.1 向柱内加入 10mL 再生缓冲液（Regeneration Buffer），进行洗涤。
4.2 加入 30mL 超纯水（ddH₂O）清洗柱体，确保将再生缓冲液彻底冲洗干净。
4.3 加入 20mL 1× 结合缓冲液（Binding buffer）洗涤，平衡填料。
4.4 加入 15mL 20% 乙醇洗涤后，再向柱内加入 5mL 20% 乙醇，盖上上盖并堵紧下方堵头，于 2-8℃条件下储存。
Protein A is a protein with a molecular weight of approximately 42 kDa, consisting of a single polypeptide chain without disulfide bonds. Its structure contains multiple highly homologous IgG-binding domains (typically 4-5), each composed of about 55-62 amino acid residues. This protein exhibits remarkable stability, retaining activity even when exposed to urea, acidic conditions (pH 2.5), or boiling. The binding range covers IgG from various mammals (e.g., humans, pigs, rabbits, mice), but the binding capacity varies among different species and IgG subclasses. For example, human IgG1, IgG2, and IgG4 subclasses form stable complexes with Protein A, while IgG3 lacks binding ability due to key site mutations in the Fc fragment; murine IgG1 shows significantly lower binding affinity to Protein A compared to subclasses such as IgG2a and IgG2b. Despite these binding differences, Protein A remains one of the most widely used ligands in antibody affinity purification, thanks to its high specific binding to IgG from most commonly used model organisms.
The Protein A Antibody Purification Kit is a standardized experimental tool designed for routine antibody purification, enabling efficient and reproducible isolation of IgG. The kit works by conjugating high-purity Protein A to highly cross-linked Polystyrene-Divinylbenzene (PS-DVB) microspheres and is suitable for purifying IgG fractions from hybridoma cell supernatants.
Product Components
Components | 2mL | 10mL | Storage | Quantity Per Test | |
P1455932A | Binding buffer(10×) | 50mL | 250mL | 2-8℃ | 5mL |
P1455932B | Elution buffer(5×) | 10mL | 50mL | 2-8℃ | 1mL |
P1455932C | Neutralizer Buffer | 10mL | 50mL | 2-8℃ | 1mL |
P1455932D | Protein A resin | 2mL | 10mL | 2-8℃ | 2mL |
P1455932E | Purification Column | 1个 | 5EA | 2-8℃ | 1EA |
P1455932F | Coomassie Brilliant Blue Staining Solution | 10mL | 50mL | 2-8℃ | 1mL |
P1455932G | Regeneration Buffer | 10mL | 50mL | 2-8℃ | 10mL (Optional) |
Note: The recommended amount of Protein A resin for each test is 2 mL.
Instructions for Use
Prior to use, dilute all component buffers to 1× concentration with deionized water. Specific operations can be adjusted according to the requirements of different purification tools.
I. Sample Preparation
Before purification, clarify hybridoma supernatants, serum, or ascites samples by centrifugation or filtration through a 0.45 μm filter to avoid clogging the purification column and reducing its lifespan.
Note: The dynamic binding capacity (DBC) of the Protein A resin provided in this kit is ≥30 mg hIgG/mL. To avoid overloading the resin and ensure purification efficiency, it is recommended that the actual IgG loading amount should not exceed 80% of this DBC (i.e., the total IgG load per column per run should be ≤24 mg). Due to variations in the expression characteristics of hybridoma cell clones, the antibody concentration in the tissue culture supernatant can fluctuate significantly. Therefore, the typical yield per purification run generally ranges between 2 mg and 10 mg. The specific yield should be determined based on actual experimental conditions, such as the antibody concentration in the supernatant and the sample volume.
II. Sample Purification
1. Column Packing
1.1 Fix the purification column vertically and ensure the bottom frit is securely attached horizontally.
1.2 Add 2 mL of Protein A Resin to the column tube. After the resin settles at the bottom, carefully insert the top frit and compact the resin gently.
1.3 Remove the bottom cap and allow the liquid in the column to drain naturally. The final effective volume of the Protein A purification column is 1 mL.
Notes: The Protein A Resin contains 50% protective solution; mix thoroughly before packing to avoid affecting purification efficiency.
When placing the frit, maintain a flat liquid level in the column to prevent air bubbles between the resin and the frit. Bubbles will reduce separation efficiency and increase the pressure required to maintain a stable flow rate.
2. Equilibration
Fill the purification column with 1× Binding Buffer (approximately 15 column volumes) to equilibrate the column, allowing the liquid to drain naturally.
3. Sample Loading
Remove the top cap of the purification column and add the clarified sample to the Protein A purification column, allowing the liquid to drain naturally.
Note: If the antibody concentration in the sample is low or the sample volume is large, increase the number of loading cycles or extend the loading time to improve the binding efficiency between the antibody and the resin.
4. Washing
Fill the purification column with 1× Binding Buffer (approximately 15 column volumes) to wash away unbound impurities, allowing the liquid to drain naturally.
5. Elution
5.1 Prepare several 1.5 mL centrifuge tubes and pre-add 10 μL of Neutralizer Buffer to each tube.
5.2 Add 3 mL of 1× Elution Buffer (approximately 3 column volumes) to each purification column and collect the eluate by gravity. Collect 8 drops (approximately 0.5 mL) per tube and repeat until elution is complete.
6. Dialysis
6.1 Take 2.5 μL of eluate from each tube, mix with 10 μL of Coomassie Brilliant Blue Staining Solution, and observe the gradient change of antibody elution.
6.2 Collect the eluate tubes with high antibody concentration and dialyze against an appropriate buffer to obtain highly purified IgG.
III. Storage of Purification Column
1. After antibody elution, add an additional 2 mL of 1× Elution Buffer to the column to wash away any residual antibody.
2. Fill the purification column with 1× Binding Buffer (approximately 15 column volumes) to equilibrate the column, allowing the liquid to drain naturally.
3. Fill the purification column with 20% ethanol (approximately 15 column volumes) for preservation, allowing the liquid to drain naturally.
4. Add 5 mL of 20% ethanol to the column, cap the top, and seal the bottom cap. Store at 2-8℃ for direct use in subsequent purifications.
IV. Column Regeneration (Optional)
Perform regeneration when a measurable decrease in column performance is observed, following these steps:
1. Add 10 mL of Regeneration Buffer to the column for washing.
2. Add 30 mL of double-distilled water (ddH₂O) to rinse the column thoroughly and remove residual Regeneration Buffer.
3. Add 20 mL of 1× Binding Buffer to wash and equilibrate the resin.
4. Add 15 mL of 20% ethanol for washing, then add 5 mL of 20% ethanol to the column. Cap the top and seal the bottom cap, and store at 2-8℃
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