基本描述

别名

Anti-RFP亲和凝胶 | RFP抗体亲和凝胶 | Anti-RFP tag亲和树脂

英文别名

Anti-RFP resin | Anti-RFP Affinity Gel | Anti-RFP Affinity Beads | Anti-RFP resin 4FF

规格或纯度

BioReagent, 50% v/v

稳定性与储存

Store at 2-8℃ long term (24 months). Do not freeze.

英文名称

Anti-RFP Agarose Resin

储存温度

2-8°C储存,禁止冷冻

运输条件

冰袋运输

产品介绍

储存缓冲液： Buffer contains 0.1% ProClin 300

红色荧光蛋白（Red Fluorecent Protein，RFP）是从海葵中分离得到的一种分子标记，常用于研究蛋白质的定位、报告基因表达和蛋白蛋白质相互作用。Anti-RFP 亲和层析介质是一种偶联了重组的抗RFP纳米抗体的琼脂糖填料，可以用于天然RFP、RFP突变体及其融合蛋白的免疫沉淀、免疫共沉淀、亲和层析等实验。

本产品具有以下特点：

(1) 使用的抗体和RFP之间具有极高的亲和力, 可以高效捕获裂解上清中的RFP标签蛋白；

(2) 免疫沉淀后，SDS-PAGE检测，背景更干净，更有利于鉴定蛋白相互作用。

(3) 琼脂糖基材具有非特异性吸附低，兼容性好的特点，每毫升载量大于1mg（最高可达到5mg/mL RFP结合载量），可以用于RFP融合蛋白的大量纯化。

阿拉丁Anti-RFP 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-RFP Nanobody
粒径范围	45~165 μm
结合能力	>1mg RFP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-RFP 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-RFP 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-RFP 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-RFP 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-RFP 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-RFP 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

Red Fluorescent Protein (RFP), isolated from sea anemones, is a molecular tag commonly used for studying protein localization, reporter gene expression, and protein-protein interactions. Anti-RFP Agarose Resin is an agarose-based matrix coupled with a recombinant anti-RFP nanobody. It can be used for immunoprecipitation (IP), co-immunoprecipitation (Co-IP), and affinity chromatography of native RFP, RFP mutants, and their fusion proteins.

This product features:

(1) The antibody exhibits extremely high affinity for RFP, enabling efficient capture of RFP-tagged proteins from lysates.
(2) Cleaner backgrounds in SDS-PAGE detection after immunoprecipitation, which is more conducive to identifying protein interactions.
(3) The agarose base matrix offers low non-specific adsorption and good compatibility. With a binding capacity greater than 1 mg per mL medium (can reach up to 5 mg/mL for RFP), it is suitable for large-scale purification of RFP fusion proteins.

Aladdin Anti-RFP 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-RFP Nanobody
Particle Size Range	45~165 μm
Binding Capacity	>1 mg RFP-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 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-RFP 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-RFP 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-RFP 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-RFP 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

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

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技术文档和文章

a1492562_anti_rfp 亲和层析介质产品说明书.pdf

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Anti-RFP 亲和层析介质

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