计算溶液所需的质量、体积或浓度。
Protein G 抗体纯化试剂盒
有货
概述
链状球菌蛋白G，简称Protein G,是一种来自链状球菌的细胞壁蛋白。分子量约为25kDa。由白蛋白结合域 (ABD1, ABD2, ABD3)和免疫球蛋白结合域构成。与Protein A相似，Protein G可与IgG的Fc区特异性结合,但Protein G能够特异性结合的抗体种类更加广泛，对于非常见物种IgG的结合能力也比较好，血清蛋白结合水平更低，纯度也更高。Protein A 和Protein G结合性能不太一样，相比Protein A，Protein G对牛、羊、马等多克隆抗体有更强的结合力，它还可以结合不能与Protein A很好结合的大鼠lgG、人lgG3和小鼠lgG1。蛋白G作为亲和配基被偶联到琼脂糖基质上，可以特异性的和样品中的抗体分子结合，而使其他杂蛋白流穿，具有极高的选择性，一步亲和层析就可达到超过95%的纯度。
本试剂盒可以纯化来自腹水，血清，细胞培养物上清等样本形式的抗体。试剂盒中的Protein G纯化填料可以重复使用10次以上，偶联在琼脂糖珠上的Protein G是纯化人、大鼠、小鼠，山羊，绵羊IgG抗体（包含单克隆或多克隆）的理想选择，高特异性，高稳定性，高亲和力，低配基脱落。
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 G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 G 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 G 纯化柱的有效体积为 1mL。
注：Protein G 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，使液体自然滴落。
2.2 上样
取下纯化柱上盖，将澄清后的样品加入 Protein G 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。
Streptococcal protein G, abbreviated as Protein G, is a cell wall protein derived from streptococci, with a molecular weight of approximately 25 kDa. It consists of albumin-binding domains (ABD1, ABD2, ABD3) and immunoglobulin-binding domains. Similar to Protein A, Protein G can specifically bind to the Fc region of IgG; however, Protein G exhibits a broader spectrum of antibody-binding specificity, with superior binding capacity for IgG from non-common species, lower serum protein binding levels, and higher purity.Protein A and Protein G differ in their binding properties. Compared with Protein A, Protein G shows stronger affinity for polyclonal antibodies from cattle, sheep, horses and other species. It can also bind to rat IgG, human IgG3 and mouse IgG1—antibody subtypes that cannot be effectively bound by Protein A.When used as an affinity ligand and conjugated to a Sepharose matrix, Protein G can specifically bind to antibody molecules in samples while allowing other impurity proteins to flow through, achieving extremely high selectivity. A single step of affinity chromatography can yield antibody purity of over 95%.
This kit is capable of purifying antibodies from various sample types, including ascites, serum, and cell culture supernatants. The Protein G purification filler in the kit can be reused more than 10 times. Protein G conjugated to Sepharose beads serves as an ideal choice for purifying monoclonal or polyclonal IgG antibodies from humans, rats, mice, goats and sheep, featuring high specificity, high stability, high affinity, and low ligand leaching.
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 G 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 G 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 G 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 G 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℃
规格
别名
蛋白 G IgG 纯化试剂盒 | 抗体纯化试剂盒(蛋白G) | 抗体Purification试剂盒(ProteinG) | NAb 蛋白 G 离心试剂盒
英文别名
Protein G IgG Purification Kit | Antibody Purification Kit (Protein G)
规格或纯度
BioReagent,纯化级
英文名称
Protein G Antibody Purification Kit
应用
抗体纯化
储存与运输
储存条件
2-8°C储存
运输条件
低温运输
稳定性与储存
各组分在相应的储存条件下保质期1年。
组分与储存
P1455933 | Components | 2mL | 10mL | Storage | Quantity Per Test |
P1455933A | 结合缓冲液 (10×) | 50mL | 250mL | 2-8℃ | 5mL |
P1455933B | 洗脱缓冲液 (5×) | 10mL | 50mL | 2-8℃ | 1mL |
P1455933C | 中和缓冲液 | 10mL | 50mL | 2-8℃ | 1mL |
P1455933D | 蛋白G填料 | 2mL | 10mL | 2-8℃ | 2mL |
P1455933E | 重力柱 | 1EA | 5*1EA | 2-8℃ | 1EA |
P1455933F | 考马斯亮蓝染色液 | 10mL | 50mL | 2-8℃ | 1mL |
P1455933G | 再生缓冲液 | 10mL | 50mL | 2-8℃ | 10mL (Optional) |
注：每个Test推荐Protein G resin使用量为2mL
高级数据
质检证书(CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
技术文档和文章
溶液计算器
常见问题
什么是「纯化级」？
「纯化级」是一个明确定义的分析纯度级别，适用于通用分析和试剂应用。它规定了最低含量百分比、对常见杂质（水分、重金属、灼烧残渣）的受控限值，以及批间一致性。「纯化级」是几个纯度级别之一——请根据方法的灵敏度和监管要求进行选择。
什么是「BioReagent」？
「BioReagent」表示该产品经过加工和检测，适用于生物和生化应用。规格可能包括内毒素限值、微生物控制、无菌性、低 DNase/RNase/蛋白酶活性，以及在相关情况下的生物活性验证。
本产品应如何储存？
请于2–8 °C条件下保存。需冷藏保存以维持规定的有效期。
本产品如何运输？
本产品采用湿冰冷藏运输。收货后请立即拆包，并按上述储存条件保存。
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