储存缓冲液：0.01M PBS and 0.02% ProClin 300，pH 7.2
阿拉丁生产的UltraBio™ Anti-DYKDDDDK琼脂糖凝胶(UltraBio™ Anti-DYKDDDDK Agarose琼脂糖凝胶)，由高质量的鼠源抗DYKDDDDK (Flag) 单克隆抗体与4%琼脂糖凝胶共价偶联制成。DYKDDDDK标签，也称为FLAG标签，是一种短肽序列，常用作蛋白标签，以方便蛋白质的纯化、检测和定位。它通常融合在目标蛋白的N端或C端，从而能够使用抗FLAG抗体轻松鉴定和分离该蛋白。因此，本产品可特异性地与动植物或者微生物裂解液、血清、腹水等样品中含有DYKDDDDK标签的蛋白结合，从而用于带有DYKDDDDK标签的融合蛋白或蛋白复合物的免疫沉淀(Immunoprecipitation, IP)或免疫共沉淀(Co-IP)。
阿拉丁UltraBio™ Anti-DYKDDDDK琼脂糖凝胶储存在含0.02% ProClin 300的PBS中，凝胶和保护液的体积比为1：1（即50%凝胶悬液，每毫升本产品中共含有0.5ml凝胶沉淀物），我司产品规格为总体积。产品信息见下表：
参数 | 指标 | 基质 | 4%琼脂糖微球 | 配基 | Recombinant DYKDDDDK tag (Binds to Flag tag) Antibody | 抗体亚型 | Mouse IgG2a | 粒径范围 | 45~165 μm | 抗体浓度 | 约3 mg DYKDDDDK tag Antibody/mL介质 | 最大压力 | 0.3 MPa，3 bar | 储存 | 0.01M PBS and 0.02% ProClin 300，pH 7.2；2-8℃ | 保质期 | 1年 |
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使用说明
1、样品准备
1.1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。
1.2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。
1.3 选择合适的裂解液，用于制备细胞或组织的裂解液。优先推荐选择阿拉丁生产的C301898 Western及IP细胞裂解液用于细胞或组织样品的裂解。根据特定的实验目的，如有必要，也可以使用阿拉丁生产的P0013B RIPA裂解液(强)、P0013C RIPA裂解液(中)或P0013D RIPA裂解液(弱)用于样品的制备。如果使用自行配制的或其它公司生产的裂解液，需要确保裂解液的pH为6-8。
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：0.01M PBS，pH7.4。
洗杂液：0.01M PBS，0.05% Tween-20，pH7.4。
3、免疫沉淀操作流程
(1) 填料准备：取40µL的Anti-DYKDDDDK琼脂糖凝胶(凝胶实际体积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300 µL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，室温反应3h。5000 × g离心1min，吸弃上清。
(4) 洗杂：加入0.5 mL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)变性洗脱：加入100 µL 1 × Loading Buffer，95℃加热5min。5000 × g离心1min，吸取上清进行SDS-PAGE电泳检测。
注意：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-DYKDDDDK琼脂糖凝胶不能重复使用。
注意事项
1. 本产品使用前一定要充分重悬，即充分颠倒若干次使混合均匀。
2. 在免疫沉淀时，建议设置阳性和阴性对照组。
3. 蛋白样品收集后宜尽快完成纯化工作，并应始终放置在4℃或冰浴，以减缓蛋白降解或变性。
4. 高浓度的DTT、巯基乙醇、盐酸胍等对本产品的非特异性结合可能有一定影响，但Western及IP细胞裂解液、RIPA裂解液或NP-40裂解液等都完全适用。
5. 为有效抑制蛋白降解，可以在蛋白样品中添加适量的蛋白酶抑制剂混合物。
6. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
7. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Aladdin's UltraBio™ Anti-DYKDDDDK Agarose is produced by high-quality mouse anti-DYKDDDDK (Flag) monoclonal antibody conjugated to 4% agarose gel. DYKDDDDK tag, also known as FLAG tag, is a short peptide sequence that is commonly used as a protein tag to facilitate protein purification, detection, and localization. It is often fused to the N- or C-terminus of a protein of interest, allowing for easy identification and isolation of the protein using anti-FLAG antibodies. Therefore, this agarose can specifically bind DYKDDDDK fusion proteins in lysates, serum, ascites, or other samples from animals, plants, or microorganisms. This product is mainly used for immunoprecipitation (IP), co-immunoprecipitation (Co-IP), or purification of DYKDDDDK-tagged proteins or their protein complexes.
Aladdin UltraBio™ Anti-DYKDDDDK Agarose is stored in PBS containing 0.02% ProClin 300, with a settled gel to storage solution ratio of 1:1(This product is a 50% gel suspension, containing 0.5 ml of settled agarose per milliliter. Our product specifications are based on total volume. Product information is detailed in the table below:
Parameter | Value/Description | Matrix | 4% Agarose Microspheres | Ligand | Recombinant DYKDDDDK tag (Binds to Flag tag) Antibody | Isotype | Mouse IgG2a | Particle Size Range | 45~165 μm | Antibody concentration | Approximately 3 mg DYKDDDDK tag Antibady /mL Settled Agarose | Maximum Pressure | 0.3 MPa，3 bar | Storage Conditions | PBS contains 0.02% ProClin 300, 2-8℃ | Shelf Life | 1 year |
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Instruction for use
1. Sample Preparation
1.1 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.
1.2 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.
1.3 Preparation of protein sample. Lysis cells or tissues with appropriate lysis buffer. The following lysis buffers are recommended for preparing lysates from cells or tissues. For optimal results, Aladdin product C301898 Cell Total Protein Lysate for Western Blotting & Immunoprecipitation is the preferred choice for lysis of cell or tissue samples. Depending on the specific experimental needs, if necessary, Aladdin products P0013B RIPA Lysis Buffer (Strong), P0013C RIPA Lysis Buffer (Medium), or P0013D RIPA Lysis Buffer (Weak) may also be used for sample preparation. If using a self-prepared lysis buffer or one from another manufacturer, ensure that the pH of the lysis buffer is between 6 and 8. when using a self-prepared lysis buffer or one produced by other companies, ensure that the pH of the lysis buffer is 6-8.
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: 0.01M PBS，pH7.4.
Wash Buffer: 0.01M PBS，0.05% Tween-20，pH7.4.
3. Immunoprecipitation (IP) Procedure
3.1 Resin Preparation: Add 40 µL of Anti-DYKDDDDK Agarose suspension (20 µL settled agarose) to a 1.5 mL tube. Centrifuge at 5000 × g for 1 minute. Discard the supernatant.
3.2 Add 0.5 mL of Equilibration Buffer to resuspend the agarose (Place the agarose in the same buffer system as the target protein to protect the protein.). Centrifuge at 5000 × g for 1 minute. Discard the supernatant. Repeat this wash step once.
3.3 Add 300 µL of sample lysate to the prepared agarose from step 3.2. Mix and incubate on a tube rotator at room temperature for 3 hours. Centrifuge at 5000 × g for 1 minute. Discard the supernatant.
3.4 Washing: Add 0.5 mL of Wash Buffer, resuspend the agarose, and mix gently. Centrifuge at 5000 × g for 1 minute. Discard the supernatant. Repeat this wash step three more times. Ensure the removal of non-specifically adsorbed substances.
3.5 Denaturing Elution (SDS-PAGE): Add 100 µL of 1 × Loading Buffer to the agarose. Heat at 95°C for 5 minutes. Centrifuge at 5000 × g for 1 minute, and load the supernatant directly onto an SDS-PAGE gel for analysis.
Note: The Standard loading buffer used in laboratories contains β-mercaptoethanol and DTT, which can break the disulfide bonds between the antibody heavy and light chains on the agarose. This method denatures the antibody, rendering the resin unusable for reuse.
Storage
Store at 4℃ for up to 12 months. Do not store at -20℃ or lower temperatures.
Matters needing attention
1. This product must be fully resuspended by inverting the tube several times prior to use.
2. For immunoprecipitation assays, it is recommended to include both positive and negative controls.
3. Protein samples should be purified as soon as possible after collection and should always be placed at 4℃ or on ice to minimize protein degradation or denaturation.
4. The non-specific binding capability of this product can be affected by high concentrations of DTT, mercaptoethanol, and guanidine hydrochloride, but it is applicable in Cell lysis buffer for Western and IP , RIPA Lysis Buffer , and NP-40 Lysis Buffer .
5. To effectively inhibit protein degradation, an appropriate amount of protease inhibitor cocktail can be added to the protein sample.
6. This product is for R&D only. Not for drug, household, or other uses.
7. For your safety and health, please wear a lab coat and disposable gloves during the operation.
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