本产品可以检测原核、真核表达的c-Myc标签融合蛋白。c-Myc多肽是由人类染色体8q24上的c-Myc基因编码，对应于人类P62的410-419（EQKLISEEDL）氨基酸。Anti-c-Myc 亲和层析介质可以识别C端，N端或内部c-Myc标签蛋白融合蛋白，可应用在Western-blot杂交技术、免疫沉淀和流式细胞计量术中, 用于检测重组蛋白质在靶细胞中的表达。
阿拉丁Anti-c-Myc 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-c-Myc 人单克隆抗体
|
粒径范围
| 45~165 μm |
| 结合能力 | ~1mg c-Myc 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 c-Myc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-c-Myc 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-c-Myc 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10-50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-c-Myc 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-c-Myc 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-c-Myc 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) 填料平衡：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) 样品结合：加入200-1000µL样品裂解液或细胞抽提物到处理好的填料中，混合均匀，在室温下置于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-c-Myc 抗体变性，洗脱后的Anti-c-Myc 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1 min，吸取上清SDS-PAGE电泳检测。
问题及解决方案
This product is designed for the detection of c-Myc tag fusion proteins expressed in both prokaryotic and eukaryotic systems. The c-Myc peptide is encoded by the c-Myc gene on human chromosome 8q24 and corresponds to amino acids 410-419 (EQKLISEEDL) of human P62. The Anti-c-Myc Agarose Resin can recognize C-terminal, N-terminal, or internal c-Myc tag fusion proteins. It is applicable in Western blotting, immunoprecipitation, and flow cytometry for detecting the expression of recombinant proteins in target cells.
Aladdin Anti-c-Myc 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-c-Myc Human Monoclonal Antibody
|
Particle Size Range
| 45~165 μm |
| Binding Capacity | ~1 mg c-Myc 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
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
Competitive Elution Buffer: 50 mM Tris, 0.15 M NaCl, 100-500 μg c-Myc 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-c-Myc Agarose Resin into a suitable chromatography column. Equilibrate with 5 column volumes (CV) of equilibration buffer to bring the medium into the same buffer system as the target protein.
(2) Apply the sample to the equilibrated Anti-c-Myc Agarose Resin. Collect the flow-through. The sample can be reloaded to increase binding efficiency.
(3) Wash with 10-50 CV of wash buffer to remove non-specifically adsorbed impurities. Collect the wash fraction.
(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 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-c-Myc Agarose Resin in the elution buffer for more than 20 minutes.*
* B. Competitive Elution: Elute with 5 CV of competitive elution buffer. Collect fractions separately.
(5) Regenerate the column with 3 CV of elution buffer, then re-equilibrate with equilibration buffer until neutral pH is reached.
(6) Store the medium in storage buffer at 2-8°C.
3.2 Batch/Binding Adsorption
(1) Medium Preparation: Transfer an appropriate amount of Anti-c-Myc 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). Ensure thorough mixing of the medium and sample solution.
(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 either the acidic or competitive elution buffer, as described in section 3.1 (4).
(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-c-Myc 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 (this brings it into the same buffer system as the target protein, protecting the protein). Centrifuge at 5000 × g for 1 min. Discard the supernatant. Repeat this step once.
(3) Sample Binding: Add 200-1000 µL of sample lysate or cell extract to the prepared medium. Mix thoroughly. Incubate the tube at room temperature on a rotator/mixer for gentle inversion for at least 1 hour to ensure sufficient contact and adsorption. Centrifuge at 5000 × g for 1 min. Discard the supernatant.
(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 to ensure removal of non-specifically bound material.
(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. Competitive Elution: Add 100 µL of competitive elution buffer to resuspend the medium. Incubate at room temperature for 30 min. Centrifuge at 5000 × g for 1 min. Carefully collect the supernatant without disturbing the medium. Store eluted samples at 4°C; for long-term storage, keep at -20°C.
* C. 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 Anti-c-Myc antibody, 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 Guide
ZJ25F1129371
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