BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
储存缓冲液： Buffer contains 0.1% ProClin 300
荧光蛋白被广泛应用于检测基因表达效率以及目的蛋白的表达和分布。YFP（Yellow Fluorescent Protein）即黄色荧光蛋白，是绿色荧光蛋白（GFP）的一种突变体，其荧光向红色光谱偏移，主要是因为GFP蛋白第203位苏氨酸被酪氨酸取代，从而发出波长较长的黄色荧光。荧光蛋白作为标签，其融合目的蛋白自发荧光，不需要目的基因的抗体或杂交就能知道目的基因在细胞中的定位，受其它物质干扰小。Anti-YFP 亲和层析介质是以重组的抗YFP纳米抗体为亲和配体，可以富集和纯化YFP及其融合表达蛋白，此外还能结合和纯化GFP和EGFP标签蛋白，不结合BFP以及RFP标签蛋白。
阿拉丁Anti-YFP 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 | 高度交联的4%琼脂糖微球 |
| 配基 | Anti-YFP Nanobody |
| 粒径范围 | 45~165 μm |
| 结合能力 | >1mg YFP标签蛋白/mL介质 |
| 最大压力 | 0.3 MPa，3 bar |
| 试剂耐受 | Stable up to 80℃, 1mM DTT, 3M Guanidinium•HCl, 8M Urea, 2M NaCl, 2% Nonidet P40 Substitute, 1% SDS, 1% Triton X-100 |
| 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
中和液：1M Tris-HCl，pH8.0
3、样品纯化
3.1 柱层析
(1) 将Anti-YFP 亲和层析介质装入合适的层析柱，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-YFP 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) 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-YFP 亲和层析介质在洗脱液中不要超过20min。
(5) 使用3倍柱体积的洗脱液清洗，然后用平衡液平衡至中性。
(6) 使用3倍柱体积的储存缓冲液平衡，2-8℃保存。
3.2 静态吸附
(1) 填料准备：取适量的Anti-YFP 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) 用酸性洗脱液洗脱。
(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-YFP 亲和层析介质 (柱体积20µL) 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1小时（对于易降解的蛋白，建议使用蛋白酶抑制剂，并在2-8℃层析柜中操作，冷库亦可）。5000×g离心1min，吸弃上清，小心不要吸走填料。
(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的酸性洗脱液，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 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介质配体变性，洗脱后的Anti-YFP 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，吸取上清进行SDS-PAGE电泳检测。
问题及解决方案
Fluorescent proteins are widely used to monitor gene expression efficiency and the expression and localization of target proteins. Yellow Fluorescent Protein (YFP), a mutant of Green Fluorescent Protein (GFP), exhibits a spectral shift towards red light, primarily due to the substitution of threonine at position 203 with tyrosine in GFP, resulting in emission of longer-wavelength yellow fluorescence. As a fusion tag, the fluorescent protein allows the fused target protein to fluoresce spontaneously, enabling the determination of the subcellular localization of the target gene without the need for specific antibodies or hybridization, and is subject to minimal interference from other substances. The Anti-YFP Agarose Resin utilizes a recombinant anti-YFP nanobody as the affinity ligand. It is designed for the enrichment and purification of YFP and its fusion proteins. Additionally, it can bind and purify GFP and EGFP tagged proteins, but does not bind BFP or RFP tagged proteins.
Aladdin Anti-YFP 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 | Specification |
| Matrix | Highly cross-linked 4% Agarose Microspheres |
| Ligand | Anti-YFP Nanobody |
| Particle Size Range | 45~165 μm |
| Binding Capacity | >1 mg YFP-tagged protein / mL Resin |
| Max Pressure | 0.3 MPa, 3 bar |
| Reagent Tolerance | Stable up to 80℃, 1mM DTT, 3M Guanidinium•HCl, 8M Urea, 2M NaCl, 2% Nonidet P40 Substitute, 1% SDS, 1% Triton X-100 |
| Storage | 0.1% ProClin 300, 2-8℃ |
| Shelf Life | 2 years |
Instructions for Use
1. Sample Preparation
Prior to column loading, ensure the sample solution has appropriate ionic strength and pH. The sample or cell culture medium can be diluted with equilibration buffer or dialyzed against it.
It is recommended to centrifuge the sample or filter it through a 0.22 μm or 0.45 μm membrane before application to reduce impurities, improve protein purification efficiency, and prevent column clogging.
2. Buffer Preparation
It is recommended to filter all 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
Neutralization Buffer: 1 M Tris-HCl, pH 8.0
3. Sample Purification
3.1 Column Chromatography
(1) Pack the Anti-YFP Agarose Resin into a suitable chromatography column. Equilibrate with 5 column volumes (CV) of equilibration buffer.
(2) Apply the sample to the equilibrated medium. 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 adsorbed impurities. Collect the wash fraction.
(4) 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 the collected fractions to adjust the pH to 7.0-8.0. Collect fractions separately.
*Note: After acidic elution, immediately re-equilibrate the medium with equilibration buffer. Do not leave the Anti-YFP Agarose Resin in the elution buffer for more than 20 minutes.*
(5) Wash the column with 3 CV of elution buffer, then re-equilibrate with equilibration buffer until neutral pH is reached.
(6) Equilibrate with 3 CV of storage buffer. Store the medium at 2-8°C.
3.2 Batch/Binding Adsorption
(1) Medium Preparation: Transfer an appropriate amount of Anti-YFP Agarose Resin to a column or tube and allow the storage solution to drain. Wash with 5 CV of equilibration buffer.
(2) Add the sample solution and incubate with shaking or gentle mixing at 4°C or room temperature for at least 30 minutes (do not use a magnetic stirrer).
(3) After incubation, centrifuge the mixture (5000 × g, 1 min) or filter to collect the medium.
(4) Transfer the medium to a column. Wash with equilibration buffer until the UV baseline stabilizes.
(5) Elute using the acidic elution buffer.
(6) Medium regeneration and storage are the same as described in sections 3.1 (5) and (6).
3.3 Immunoprecipitation (IP) Protocol
(1) Medium Preparation: Add 40 µL of the Anti-YFP Agarose Resin slurry (approx. 20 µL settled medium volume) to a 1.5 mL microcentrifuge tube. Centrifuge at 5000 × g for 1 min. Carefully remove and discard the supernatant.
(2) Medium Equilibration: Add 0.5 mL of equilibration buffer to resuspend the medium. Centrifuge at 5000 × g for 1 min. Discard the supernatant. Repeat this step once.
(3) Sample Binding: Add 200-1000 µL of sample to the prepared medium. Mix thoroughly. Incubate the tube at room temperature on a rotator/mixer for gentle inversion for at least 1 hour (For proteins prone to degradation, the use of protease inhibitors and performing the procedure at 2-8°C in a cold room or chromatography refrigerator is recommended). Centrifuge at 5000 × g for 1 min. Discard the supernatant, being careful not to disturb the medium pellet.
(4) Washing: Add 0.5 mL of wash buffer to resuspend the medium. Mix gently. Centrifuge at 5000 × g for 1 min. Discard the supernatant. Repeat this washing step three more times.
(5) Sample Elution: Choose the elution method based on downstream application needs.
* A. Acidic Elution: Add 100 µL of acidic elution buffer to resuspend the medium. Incubate at room temperature for 5 min. Centrifuge at 5000 × g for 1 min. Carefully collect the supernatant without disturbing the medium. Neutralize the eluate immediately using neutralization buffer. Store eluted samples at 4°C; for long-term storage, keep at -20°C.
* B. Denaturing Elution: Conventional protein loading buffer contains β-mercaptoethanol or DTT, which reduces disulfide bonds, and SDS, which denatures proteins. This method will elute the target protein but also denature the ligand (nanobody), making the medium unsuitable for reuse.
Add 20 µL of 2× Loading Buffer to the medium. Heat at 95°C for 5 min. Centrifuge at 5000 × g for 1 min. Carefully collect the supernatant for SDS-PAGE analysis.
Troubleshooting
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F1029201 |
分析证书 | A1492563 |
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