HA 标签是人流感血凝素的第98-106氨基酸序列YPYDVPDYA，对外源靶蛋白的空间结构影响小, 容易构建成标签蛋白融合到N端或者C端，因此常被用于重组蛋白的融合表达。Anti-HA 亲和层析介质以4%琼脂糖凝胶为基质，杂蛋白非特异性结合少，可用于HA标签融合蛋白的纯化和免疫沉淀（IP）。
阿拉丁Anti-HA 亲和层析介质储存在含0.1% ProClin 300的保护液中，凝胶和保护液的体积比为1：1，我司产品规格为实际凝胶的体积。
参数
| 指标
|
基质
| 4%琼脂糖微球
|
配基
| Anti-HA 鼠单克隆抗体
|
粒径范围
| 45~165 μm |
| 结合能力 | >1mg HA标签蛋白/mL介质
|
最大压力
| 0.1 MPa，1 bar |
储存
| 0.1% ProClin 300，2~8℃
|
保质期
| 2年 |
使用说明
1、样品准备
上柱之前要确保样品溶液有合适的离子强度和pH值，可以用平衡液对样品或细胞培养液稀释，或者用平衡液透析。
样品在上样前建议离心或用0.22μm或0.45μm滤膜过滤，减少杂质，提高蛋白纯化效率和防止堵塞柱子。
2、缓冲液的准备
所用水和缓冲液在使用之前建议用0.22μm或0.45μm滤膜过滤。
平衡液：10mM Tris，0.15M NaCl，pH7.4
洗杂液：10mM Tris，0.15M NaCl，0.05% Tween-20，pH7.4
化学洗脱液：0.1M glycine HCl, pH 2.0-2.8或3M NaSCN或者50mM NaOH
| 溶液 | 优点 | 缺点
|
0.1M glycine HCl, pH 2.0-2.8
| 如果目的蛋白在低pH下稳定，不会破坏填料结合
| 洗脱效率低
蛋白可能变性
|
3M NaSCN
| 洗脱效率高，不会破坏填料结合能力
| 蛋白可能变性
|
50mM NaOH
| 洗脱效率高
| 蛋白可能变性
减少填料使用寿命 |
竞争性洗脱液：50mM Tris，0.15M NaCl，100-500µg HA多肽/mL，pH7.4
中和液：1M Tris-HCl，pH8.5
3、样品纯化
3.1 柱层析
(1) 将Anti-HA 亲和层析介质装入合适的层析柱，层析用5倍柱体积的平衡液进行平衡，使填料处于与目的蛋白相同的缓冲体系下。
(2) 将样品加到平衡好的Anti-HA 亲和层析介质中，收集流出液，可以反复上样增加结合效率。
(3) 用10-20倍柱体积的洗杂液进行清洗，去除非特异性吸附的杂蛋白，收集洗杂液。
(4)
样品洗脱
A 酸性洗脱：使用5倍柱体积的酸性洗脱液洗脱，向洗脱组分中加入洗脱体积十分之一的中和液，调节pH值至7.0-8.0，分管收集。
注：酸性洗脱后填料要立即用平衡液平衡，Anti-HA 亲和层析介质在洗脱液中不要超过20min。
B 化学洗脱：使用5倍柱体积的化学洗脱液洗脱，分管收集。 注：化学洗脱后填料要立即用平衡液平衡，Anti-HA 亲和层析介质在洗脱液中不要超过20min。
C 竞争性洗脱：使用5倍柱体积的竞争性洗脱液洗脱。分管收集。
(5) 使用3倍柱体积的化学洗脱液（甘氨酸洗脱液）再生，然后用平衡液平衡至中性。
(6) 然后保存在储存缓冲液中，2-8℃保存。
3.2 静态吸附
(1) 填料准备：取适量的Anti-HA 亲和层析介质加入层析柱中，流干保护液。加入5倍柱体积的平衡液清洗。
(2) 加入样品溶液，4℃或室温震荡孵育30min（不能磁力搅拌），确保填料与样品溶液充分混合。
(3) 孵育完毕后，将填料混合液离心（5000×g离心1min）或过滤收集填料。
(4) 将填料装入层析柱中，用平衡液清洗直至紫外稳定。
(5) 用化学洗脱液或竞争性洗脱液洗脱，参考3.1中(4)。
(6) 填料再生和保存参考3.1中(5)和(6)。
3.3 免疫沉淀操作流程
(1) 填料准备：取40µL的Anti-HA 亲和层析介质 (柱体积20 µL) 混合液加入到1.5mL 离心管中，5000×g离心1min，吸弃上清。
(2) 填料加入0.5mL平衡液，悬浮填料（使填料处于与目的蛋白相同的缓冲体系下，起到保护蛋白的作用），5000×g离心1min，吸弃上清。重复一次。
(3) 加入200-1000µL样品裂解液到处理好的填料中，混合均匀，在室温下置于翻转混合仪轻轻翻转离心管，促使样品和填料充分接触并吸附，室温至少1h。5000×g离心1min，吸取上清，留样检测。
(4) 洗杂：加入0.5mL的洗杂液，悬浮填料，轻轻混匀，5000×g离心1min，吸弃上清。再重复三次。确保去除非特异性吸附。
(5) 样品洗脱：可根据后期检测的需要选择不同的洗脱方法。
A 化学洗脱：加入100µL的化学洗脱液（0.1M glycine HCl, pH2.0-2.8，3M NaSCN或者50 mM NaOH），悬浮填料，室温孵育5min。5000×g离心 1 min。小心取出上清，不要吸到填料，用中和液中和。洗脱样品放置4℃，长时间放置-20℃保存。
B 竞争性洗脱：加入100µL竞争性洗脱液洗脱。室温孵育30min，5000×g离心1min。小心取出上清，不要吸到填料，重复洗脱1-2次。洗脱样品放置4℃，长时间放置-20℃保存。
C 变性洗脱：实验室常规蛋白上样缓冲液（Loading Buffer）中含有β-巯基乙醇和DTT，可以使填料中抗体重链和轻链断开。含有SDS的样品缓冲液可以使Anti-HA抗体变性，洗脱后的Anti-HA 亲和层析介质没办法重复使用。
每管中加入20µL 2×Loading Buffer，95℃加热5min。5000×g离心1min，吸取上清SDS-PAGE电泳检测。
问题及解决方案
The HA tag consists of the amino acid sequence YPYDVPDYA (residues 98-106 of human influenza hemagglutinin). It has minimal impact on the tertiary structure of the target foreign protein and can be easily fused to either the N- or C-terminus, making it a popular choice for recombinant protein expression. Anti-HA Agarose Resin is based on a 4% agarose gel matrix, which minimizes non-specific binding of host cell proteins, making it suitable for both the purification and immunoprecipitation (IP) of HA-tagged fusion proteins.
Aladdin Anti-HA Agarose Resin is stored in a solution containing 0.1% ProClin 300, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Parameter
| Value / Description
|
Matrix
| 4% Agarose Microspheres
|
Ligand
| Anti-HA Mouse Monoclonal Antibody
|
Particle Size Range
| 45~165 μm |
| Binding Capacity | >1 mg HA-tagged protein / mL resin
|
Maximum Pressure
| 0.1 MPa, 1 bar |
Storage Conditions
| 0.1% ProClin 300, 2~8℃
|
Shelf Life
| 2 years |
Protocol
1. Sample Preparation
Ensure the sample solution has appropriate ionic strength and pH before loading. Dilute the sample or cell culture supernatant with equilibration buffer, or dialyze the sample against equilibration buffer.
Clarify the sample by centrifugation or filtration through a 0.22 μm or 0.45 μm membrane to reduce impurities, improve purification efficiency, and prevent column clogging.
2. Buffer Preparation
It is recommended to filter water and buffers through a 0.22 μm or 0.45 μm membrane before use.
Equilibration Buffer: 10 mM Tris, 0.15 M NaCl, pH 7.4
Wash Buffer: 10 mM Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.4
Chemical Elution Buffers:
Competitive Elution Buffer: 50 mM Tris, 0.15 M NaCl, 100–500 µg HA peptide / mL, pH 7.4
Neutralization Buffer: 1 M Tris-HCl, pH 8.5
Comparison of Chemical Elution Buffers
| Solution | Advantages | Disadvantages
|
0.1M glycine HCl, pH 2.0-2.8
| Does not damage resin binding capacity if target protein is stable at low pH
| Low elution efficiency; target protein may denature
|
3M NaSCN
| High elution efficiency; does not damage resin binding capacity
| Target protein may denature
|
50mM NaOH
| High elution efficiency
| Target protein may denature; reduces resin lifespan
|
3. Sample Purification
3.1 Column Chromatography
(1) Pack the Anti-HA Agarose Resin into a suitable chromatography column. Equilibrate the column with 5 column volumes (CV) of Equilibration Buffer.
(2) Load the sample onto the equilibrated resin. Collect the flow-through. The sample can be reloaded to increase binding efficiency.
(3) Wash with 10–20 CV of Wash Buffer to remove non-specifically bound proteins. Collect the wash fractions.
(4) Elution:
* A. Acidic Elution: Elute with 5 CV of acidic elution buffer (e.g., 0.1 M Glycine-HCl, pH 2.0–2.8). Add a volume of Neutralization Buffer equal to one-tenth of the elution volume to each fraction to adjust the pH to 7.0–8.0. Collect fractions separately.
* Note: After acidic elution, the resin must be immediately re-equilibrated. Do not expose the resin to the acidic elution buffer for more than 20 minutes.
* B. Chemical Elution: Elute with 5 CV of a chemical elution buffer (e.g., 3 M NaSCN or 50 mM NaOH). Collect fractions separately.
* Note: After chemical elution, the resin must be immediately re-equilibrated. Do not expose the resin to the chemical elution buffer for more than 20 minutes.
* C. Competitive Elution: Elute with 5 CV of Competitive Elution Buffer. Collect fractions separately.
(5) Regenerate the resin with 3 CV of a chemical elution buffer (e.g., Glycine-HCl), then re-equilibrate with Equilibration Buffer until neutral pH is reached.
(6) Store the resin in Storage Buffer at 2–8°C.
3.2 Batch/Binding Method
(1) Resin Preparation: Transfer an appropriate amount of Anti-HA Agarose Resin to a column and drain the storage solution. Wash with 5 CV of Equilibration Buffer.
(2) Add the sample solution. Incubate with shaking at 4°C or room temperature for 30 minutes (avoid magnetic stirring). Ensure thorough mixing.
(3) After incubation, centrifuge the mixture (5,000 × g, 1 min) or filter to collect the resin.
(4) Transfer the resin to a column. Wash with Equilibration Buffer until the UV baseline stabilizes.
(5) Elute using either the Chemical or Competitive Elution method as described in section 3.1 (4).
(6) Regenerate and store the resin as described in sections 3.1 (5) and (6).
3.3 Immunoprecipitation (IP) Procedure
(1) Resin Preparation: Add 40 µL of Anti-HA Agarose Resin suspension (20 µL settled resin) to a 1.5 mL tube. Centrifuge at 5,000 × g for 1 min. Discard the supernatant.
(2) Add 0.5 mL of Equilibration Buffer to resuspend the resin. Centrifuge at 5,000 × g for 1 min. Discard the supernatant. Repeat this wash step once.
(3) Add 200–1000 µL of sample lysate to the prepared resin. Mix and incubate on a tube rotator at room temperature for at least 1 hour. Centrifuge at 5,000 × g for 1 min. Collect the supernatant for analysis.
(4) Washing: Add 0.5 mL of Wash Buffer, resuspend the resin, and mix gently. Centrifuge at 5,000 × g for 1 min. Discard the supernatant. Repeat this wash step three more times.
(5) Elution: Choose the elution method based on downstream application.
* A. Chemical Elution: Add 100 µL of chemical elution buffer (0.1 M Glycine-HCl pH 2.0-2.8, 3 M NaSCN, or 50 mM NaOH) and resuspend the resin. Incubate at room temperature for 5 min. Centrifuge at 5,000 × g for 1 min. Carefully collect the supernatant and neutralize immediately if acidic. Store eluted samples at 4°C short-term or -20°C long-term.
* B. Competitive Elution: Add 100 µL of Competitive Elution Buffer and resuspend the resin. Incubate at room temperature for 30 min. Centrifuge at 5,000 × g for 1 min. Carefully collect the supernatant. Repeat elution 1-2 times. Store eluted samples at 4°C short-term or -20°C long-term.
* C. Denaturing Elution (SDS-PAGE): Add 20 µL of 2× Loading Buffer (contains SDS and reducing agents like β-mercaptoethanol/DTT) to the resin. Heat at 95°C for 5 min. Centrifuge at 5,000 × g for 1 min, and load the supernatant directly onto an SDS-PAGE gel for analysis. Note: This method denatures the antibody, rendering the resin unusable for reuse.
Troubleshooting Guide
ZJ25F1029203
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