碧云天生产的Anti-HA Affinity Gel (Anti-HA亲和凝胶)，也称Anti-HA IP Gel、Anti-HA免疫沉淀凝胶或Anti-HA琼脂糖凝胶，由高质量的单克隆鼠源HA抗体与琼脂糖共价偶联而成，可与常规蛋白表达系统(如哺乳动物细胞、细菌或酵母)中含有HA标签的蛋白结合，从而用于带有HA标签的融合蛋白或蛋白复合物的免疫沉淀(Immunoprecipitation, IP)或纯化。
HA标签(HA-tag)、Myc标签(Myc-tag)、Flag标签(Flag-tag)、His标签(His-tag)和GST标签(GST-tag)等是表达载体上最常见的一些标签，通过与这些标签的融合表达可以非常方便地检测目的蛋白及与目的蛋白相互结合的蛋白，也可以非常方便地用于目的蛋白的纯化。HA-tag是9个氨基酸残基(YPYDVPDYA)组成的多肽，源于人流行性感冒病毒血凝素(influenza virus hemagglutinin)的98-106位氨基酸序列，所以称为HA多肽。其常用的形式有HA和3X HA，通过基因重组技术把HA-tag的核酸序列与目的基因的5'端或3'端连接，就可以最终表达形成HA-tag的目的蛋白。HA-tag具有以下优点：HA-tag通常不会与目的蛋白相互作用，并且大多数情况下不会影响目的蛋白的功能；HA-tag作为标签蛋白，后续通过HA抗体(AH158/ AF2858)、Anti-HA磁珠(P2121)或Anti-HA亲和凝胶(P2287)即可对目的基因的表达、定位及功能进行检测或对目的蛋白进行纯化、免疫沉淀或免疫共沉淀等。基于以上优点，HA标签已被广泛应用于蛋白表达、纯化、鉴定、相互作用和功能等多方面的研究。
Anti-HA Affinity Gel (Anti-HA亲和凝胶)，也被称为Anti-YPYDVPDYA Affinity Gel/Beads/Resin或Anti-YPYDVPDYA IP Gel/Beads/Resin，可特异性地结合HA标签融合蛋白，广泛应用于带有HA标签的融合蛋白或蛋白复合物的免疫沉淀或纯化等实验。
本产品有较高的融合蛋白结合量、特异性强：每ml纯凝胶(settled gel)含有约7.5mg HA抗体，可结合约1.5mg融合蛋白，且特异性强，非特异的杂蛋白结合少。
本产品可结合多种形式的HA标签蛋白：本产品可特异性地结合甲硫氨酸修饰的N端HA融合蛋白(Met-HA-Protein)、N端HA融合蛋白(HA-Protein)、C端HA融合蛋白(Protein-HA)。
本产品可选择多种洗脱方法：本产品根据蛋白结构的完整性、生物功能及后续应用的要求等，可使用多种洗脱方法，包括HA多肽、酸性和SDS-PAGE上样缓冲液等洗脱液进行洗脱。特别是HA多肽洗脱后不包含抗体的轻链和重链，可以有效解决免疫沉淀后Western实验中轻链和重链的干扰问题。
本产品可重复使用多次，性价比高：在正常情况下，本产品用于相同蛋白的纯化时可回收使用3-5次。如果用于免疫共沉淀检测蛋白-蛋白的相互作用，不推荐重复使用。
本产品的主要指标如下表：
| Cat. No. | P2287 |
| Product name | Anti-HA Affinity Gel (Anti-HA亲和凝胶) |
| Product content | 50% settled gel in 50% glycerol with 10mM phosphate-buffered saline and preservative (pH7.4) |
| Matrix | 4% agarose |
| Average bead size | ~90μm |
| Antibody | Mouse monoclonal antibody against HA-tag |
| Isotype | IgG1 |
| M.W. of antibody | Approximately 150kDa |
| Antibody concentration | Approximately 7.5mg HA antibody per ml settled gel |
| Binding capacity | Approximately 1.5mg HA-tagged protein per ml settled gel |
| Elution method | Acid, alkaline, neutral, peptide competitive or SDS-PAGE loading buffer elution. Note: If elute with SDS-PAGE loading buffer, the light (~25kDa) and heavy (~50kDa) chain of antibody will be denatured and release from the gel. |
| Reagent compatibility | Chaotropic reagents will denature the target HA-tagged protein. Do not exceed 0.3M GuHCl or 1.5M Urea. |
| Application | Suitable for IP, Co-IP and protein purification. |
| Storage | -20℃ |
本产品为50%凝胶悬液，包装体积为总体积，每毫升本产品中共含有0.5ml纯凝胶(沉淀物)。
包装清单：| 产品编号 | 产品名称 | 包装 |
| P2287-0.5ml | Anti-HA Affinity Gel (Anti-HA亲和凝胶) | 0.5ml |
| P2287-2ml | Anti-HA Affinity Gel (Anti-HA亲和凝胶) | 2ml |
| P2287-10ml | Anti-HA Affinity Gel (Anti-HA亲和凝胶) | 10ml |
| — | 说明书 | 1份 |
-20℃保存，一年有效。
注意事项：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。
本产品含有微量的防腐剂，不会影响常规的蛋白或蛋白复合物的纯化和免疫沉淀。但如果后续涉及酶活性测定，使用本产品前宜先用TBS等适当溶液洗涤凝胶3次，以充分消除防腐剂可能产生的干扰。
在免疫沉淀或纯化时，建议设计阳性和阴性对照组。
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。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，例如碧云天的P1005/P1006蛋白酶抑制剂混合物(通用型)、P1048/P1049蛋白酶磷酸酶抑制剂混合物(通用型, 质谱兼容, 50X)、P1010/P1011蛋白酶抑制剂混合物(哺乳动物样品抽提用, 100X)、P1050/P1051蛋白酶磷酸酶抑制剂混合物(哺乳动物样品抽提用, 50X)等，或P1025/P1026蛋白酶抑制剂混合物(细菌抽提用)。
高浓度的DTT、巯基乙醇、盐酸胍等对本产品与标签蛋白的结合可能有一定影响，但Western及IP细胞裂解液(P0013)、RIPA裂解液(P0013B/C/D)或NP-40裂解液(P0013F)等都完全适用。碧云天生产的不同裂解液的主要特点和差异，以及如何选择裂解液可参考我们的相关网页：http://www.beyotime.com/support/lysis-buffer.htm。
若离心不能完全除去蛋白样品中的不溶物，可以将样品溶液用0.45μm的滤膜过滤。
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。
为了您的安全和健康，请穿实验服并戴一次性手套操作。
| Problem | Possible Causes | Solution |
| Large amount of tagged protein found in the flow through. | Binding time is not enough. | If using batch method, increase the binding time experimentally; If using column method, use a lower flow rate when loading samples. |
| Column is overloaded. | Reduce the amount of the sample added to the gel or increase the amount of gel. | |
| Tag is not accessible to gel. | Expose the epitope tag by adding low amount of denaturant to the protein extract (dialysis may be needed before applying onto gel), or fuse the tag to the other terminus of the target protein. | |
| Gel has not been regenerated since last purification. | Perform gel regeneration procedure prior to binding. | |
| Reagent compatibility problem. | Dialyze the sample against TBS before purification procedure. | |
| The target protein has been degraded. | 1.Prepare fresh lysates. Avoid using frozen lysates. 2.Perform purification at lower temperature, such as 4℃. 3.Use appropriate protease inhibitors in the lysate or increase their concentrations to prevent degradation of the fusion protein. |
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| Very few or no tagged protein exists in the eluate. | Protein is not completely eluted. | Change elution methods. |
| No target protein expressed. | Make sure the protein of interest contains the tag by Western blot or dot blot analyses. | |
| Very low protein expression level. | 1.Use larger volume of cell lysate. 2.Optimize expression conditions to raise the protein expression level. |
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| Washes are too stringent. | 1.Reduce the number of washes. 2.Avoid adding high concentrations of NaCl to the mixture. 3.Use solutions that contain less or no detergent |
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| Incubation times are inadequate. | Increase the incubation times with the affinity gel (from several hours to overnight). | |
| Interfering substance is present in sample. | 1.Lysates containing high concentrations of DTT, 2-mercaptoethanol, or other reducing agents may destroy antibody function, and must be avoided. 2.Excessive detergent concentrations may interfere with the antibody-antigen interaction. Detergent levels in buffers may be reduced by dilution. |
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| Detection system is inadequate. | If Western blot detection is used: 1.Check primary and secondary antibodies using proper controls to confirm binding and reactivity. 2.Verify that the transfer was adequate by staining the membrane with Ponceau S. 3.Use fresh detection substrate or try a different detection system. |
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| Multiple protein bands found in the eluate. | The protein is not stable at room temperature. | Purify the target protein at lower temperature, such as 4℃. |
| Protein degradation due to proteases activity during purification process. | Add protease inhibitors to cell lysate. | |
| Non-specific binding. | 1.Prepare cell lysate again. 2.Add additional wash steps. |
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| Background is too high. | Proteins bind nonspecifically to the monoclonal antibody, the gel beads, or the microcentrifuge tubes. | 1.Pre-clear lysate with Mouse IgG-Agarose (P2055+A7028) to remove nonspecific binding proteins. 2.After suspending beads for the final wash, transfer entire sample to a clean microcentrifuge tube before centrifugation. |
| Washes are insufficient. | 1.Increase the number of washes. 2.Prolong duration of the washes, incubating each wash for at least 15 minutes. 3.Increase the salt and/or detergent concentrations in the wash solutions. 4.Centrifuge at lower speed to avoid nonspecific trapping of denatured proteins from the lysate during the initial centrifugation of the affinity gel complexes. |
