Flag 标签是一个由八个亲水氨基酸组成的多肽片段，定位在融合蛋白表面，因此更易与抗体结合以及被肠激酶分解。Anti-Flag 亲和层析介质是以抗Flag (DYKDDDDK) 抗体为亲和配体，一步纯化原核、酵母或哺乳动物细胞表达的Flag标签融合蛋白。本产品以4%琼脂糖凝胶为基质，杂蛋白非特异性结合少，可用于Flag标签融合蛋白的纯化和免疫沉淀（IP）。
阿拉丁Anti-Flag 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-DYKDDDDK Antibody
|
粒径范围
| 45~165 μm |
| 结合能力 | >1mg DYKDDDDK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/洗杂液：50mM Tris，0.15M NaCl，pH7.4
酸性洗脱液：0.1M glycine HCl，pH3.0
竞争性洗脱液：50mM Tris，0.15M NaCl，100-500μg Flag多肽/mL，pH7.4
中和液：1M Tris-HCl，pH8.0
3、样品纯化
3.1 柱层析
(1) 将Anti-Flag 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-Flag 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-Flag 亲和层析介质在洗脱液中不要超过20min。
B 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-Flag 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℃或室温震荡孵育至少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) 用酸性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-Flag 亲和层析介质 (柱体积20 µL)混合液加入到2mL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，吸弃上清。
(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 酸性洗脱：加入100µL酸性洗脱液，悬浮填料。室温孵育5min，5000×g离心1min。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。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-DYKDDDDK抗体变性，洗脱后的Anti-Flag 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。
试剂兼容性
试剂名称
| 最大耐受浓度
| 备注
|
β-巯基乙醇
| 10 mM
| 纯化过程中应避免使用，如果在IP中使用，填料不能回收重复利用
|
DTT
| 80 mM
| 纯化过程中应避免使用，如果在IP中使用，填料不能回收重复利用
|
SDS
| --
| 纯化过程中应避免使用，如果在IP中使用，填料不能回收重复利用
|
EDTA
| 5 mM
| 过高的EDTA会降低蛋白回收率
|
Tween-20
| 5%
| 过高浓度会影响标签蛋白结合效率
|
Triton X-100
| 5%
| 过高浓度会影响标签蛋白结合效率
|
NP-40
| 4%
| 过高浓度会影响标签蛋白结合效率
|
盐酸胍
| 0.3 M
| 过高浓度会使抗体变性
|
尿素
| 1.5 M
| 过高浓度会使抗体变性
|
甘油
| 20%
| 过高浓度会影响标签蛋白结合
|
氯化钠
| 1 M
| 减少非特异性吸附 |
问题及解决方案
The Flag tag is an octapeptide composed of hydrophilic amino acids, strategically positioned on the surface of fusion proteins. This location facilitates easier binding to antibodies and cleavage by enterokinase. Anti-Flag Agarose Resin utilizes an anti-Flag (DYKDDDDK) antibody as the affinity ligand for the one-step purification of Flag-tagged fusion proteins expressed in prokaryotic, yeast, or mammalian cell systems. This product 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 Flag-tagged fusion proteins.
Aladdin Anti-Flag 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-DYKDDDDK Antibody
|
Particle Size Range
| 45~165 μm |
| Binding Capacity | >1 mg DYKDDDDK-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/Wash Buffer: 50 mM Tris, 0.15 M NaCl, pH 7.4
Acidic Elution Buffer: 0.1 M Glycine-HCl, pH 3.0
Competitive Elution Buffer: 50 mM Tris, 0.15 M NaCl, 100-500 μg Flag peptide / mL, pH 7.4
Neutralization Buffer: 1 M Tris-HCl, pH 8.0
3. Sample Purification
3.1 Column Chromatography
(1) Pack the Anti-Flag Agarose Resin into a suitable chromatography column. Equilibrate the column with 5 column volumes (CV) of Equilibration Buffer to bring the resin into the same buffer system as the target protein.
(2) Load the sample onto the equilibrated Anti-Flag Agarose Resin. Collect the flow-through. The sample can be reloaded to increase binding efficiency.
(3) Wash with 10 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. 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 Anti-Flag Agarose Resin to the acidic elution buffer for more than 20 minutes.**
* B. Competitive Elution: Elute with 5 CV of Competitive Elution Buffer. Collect fractions separately.
(5) Regenerate the resin with 3 CV of the respective Elution Buffer, 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-Flag 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 at least 30 minutes (avoid magnetic stirring). Ensure thorough mixing of the resin and sample.
(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 Acidic 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-Flag Agarose Resin suspension (20 µL settled resin) to a 2 mL tube. Centrifuge at 5,000 × g for 1 min. Carefully remove and discard the supernatant.
(2) Add 0.5 mL of Equilibration Buffer to resuspend the resin (this brings it into the correct buffer system, protecting the protein). 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 thoroughly and incubate on a tube rotator or roller mixer at room temperature for at least 1 hour to facilitate binding. Centrifuge at 5,000 × g for 1 min. Discard the supernatant.
(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 to ensure removal of non-specifically bound material.
(5) Elution: Choose the elution method based on downstream application requirements.
* A. Acidic Elution: Add 100 µL of Acidic Elution Buffer and resuspend the resin. Incubate at room temperature for 5 min. Centrifuge at 5,000 × g for 1 min. Carefully collect the supernatant without disturbing the resin. Neutralize immediately with Neutralization Buffer. 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. Store eluted samples at 4°C short-term or -20°C long-term.
* C. Denaturing Elution (SDS-PAGE): Standard protein loading buffer (containing β-mercaptoethanol/DTT and SDS) will denature the anti-Flag antibody, releasing the bound protein but rendering the resin unusable for reuse. Add 20 µL of 2× Loading Buffer 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.
Reagent Compatibility
Reagent
| Maximum Tolerant Concentration
| Notes
|
β-Mercaptoethanol
| 10 mM
| Avoid during purification; if used in IP, resin cannot be reused
|
DTT
| 80 mM
| Avoid during purification; if used in IP, resin cannot be reused
|
SDS
| --
| Avoid during purification; if used in IP, resin cannot be reused
|
EDTA
| 5 mM
| Higher concentrations reduce protein recovery
|
Tween-20
| 5%
| High concentrations may reduce binding efficiency
|
Triton X-100
| 5%
| High concentrations may reduce binding efficiency
|
NP-40
| 4%
| High concentrations may reduce binding efficiency
|
Guanidine HCl
| 0.3 M
| Higher concentrations denature the antibody
|
Urea
| 1.5 M
| Higher concentrations denature the antibody
|
Glycerol
| 20%
| High concentrations may affect protein binding
|
NaCl
| 1 M
| Helps reduce non-specific adsorption |
Troubleshooting Guide
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ZJ25F1129250
危险品化学品经营许可证（带存储）