本产品是将刀豆蛋白A（Concanavalin A，简称Con A）键合在琼脂糖凝胶微球上形成的生物亲和层析分离介质，主要用于各种带甘露醇及葡萄糖残基的糖蛋白（包括糖蛋白病毒）、多糖和糖脂的分离和纯化。Con A是从刀豆中提取出来的一种四聚体金属蛋白，是凝集素的一种，在Mn²⁺和Ca²⁺存在的情况下，可结合α-D-吡喃甘露糖基、α-D-吡喃葡萄糖基和具有C-3、C-4或C-6羟基空间相关残基的分子。Con A亲和层析介质的应用领域还包括酶-抗体偶联物的纯化、IgM的纯化、洗涤剂溶解的膜中细胞表面糖蛋白的分离纯化、膜囊泡的分离以及含碳水化合物物质组成变化的研究等。
阿拉丁Con A亲和层析介质储存在 20% (v/v) Ethanol, 0.1 M acetate, 1 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂, pH 6.0 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
| 交联琼脂糖
|
配基
| Con A，≥8 mg/mL
|
粒径范围 ①
| 45~165 μm
|
平均粒径
| ~90 μm
|
结合能力 ②
| ≥30 mg Thyroglobulin/mL
|
推荐工作流速 ③
| 15~150 cm/h
|
最大流速与压力 ④
| 900 cm/h, 0.3 MPa
|
使用pH
| pH 4~9
|
化学稳定性
| 在以下溶液中稳定：常用的水相缓冲液；应避免螯合剂EDTA、8M尿素，或pH值低于3的溶液，以免凝集素中失去金属离子而失去活性。
|
储存
| 20% (v/v) Ethanol, 0.1 M acetate, 1 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂, pH 6.0，2~8℃
|
保质期
| 3年 |
注：
① 90%体积以上的微球在此粒径范围；
② 在2 mg/mL的猪甲状腺球蛋白溶液中孵育2 h的结合量，该测试方法与动态结合载量数值接近；
③ 推荐流速是指该流速下结合效果较好，并非只能在该流速范围内工作；
④ 10cm柱高下的最大测试流速。
使用说明
1、色谱柱装填
以下阐述与层析系统连接时，填料的色谱柱装填方法。
（1）所有需要用到的材料的温度要与色谱操作的温度一样，液体最好做脱气处理。
（2）填料用量计算：通过沉降定量填料体积，需要的沉降填料体积=柱体积×压缩比（也称压缩因子）。沉降体积是指填料在20%乙醇保存液中自然沉降完全读取的稳定体积。Con A亲和层析介质的压缩比为1.15。为使达到装柱比，可通过柱头下压的方法，也可以通过高流速压柱。
（3）填料清洗：将填料悬液充分摇匀后量取相应体积，抽滤除去液体，并用约3倍填料体积的纯化水洗涤，重复3次，以除去保存液。
（4）装柱填料悬液准备：将填料转移到适当的容器中，加入适量的装柱液，制成 50~75 v%的装柱填料悬液，使用前搅匀。
（5）装填前准备：在清洗干净的层析柱下端加入装柱液，以除去下垫片及层析柱下端的空气，在柱内保留少量的蒸馏水，拧紧下堵头，调整柱子使其垂直于地面。
（6）装填：将搅匀后的填料悬液一次性缓慢倒入层析柱内（必要时使用装柱器），为避免引入气泡，应使之沿层析柱内壁自然流下。将所有填料加入后，用装柱液将装柱器加满，拧紧装柱器上盖，将柱子与层析系统连接。
（7）压柱：使柱内填料自然沉降（或10-20 cm/h的低流速下沉降），待填料沉降完全后（填料与液体的界面清晰），去除装柱器，装上上柱头，并将柱头下降至界面处； 通过高流速（推荐流速见下表，注意柱压不超过0.3 MPa），继续压柱至界面清晰稳定，标记界面稳定时的柱高。停泵，打开柱头上的阀门/堵头，关闭柱底的阀门/堵头，下压柱头至标记位置下方与压缩比对应的位置，旋紧柱头，装柱完成。装柱完成后，需用高流速平衡柱内的填料。
装柱条件
| Con A亲和层析介质
|
压缩比
| 1.15
|
装柱流速
| 300-600 cm/h
|
2、柱效测定和评价
完成装柱后、使用前可通过柱效测定和评价可以确认层析柱装填质量。柱效通常用理论塔板高度（HETP）和非对称因子（As）来评价。
柱效测定可以采用丙酮或者NaCl作为样品进行，按照下表配制样品溶液和流动相。
样品
| 1.0%丙酮
| 0.8~1.0 M NaCl
|
样品体积
| 1.0%柱体积
| 1.0%柱体积
|
流动相
| 纯水
| 0.4 M NaCl
|
流速
| 30 cm/h
| 30 cm/h
|
检测器
| UV-280 nm
| 电导
|
根据UV或者电导率曲线计算理论塔板高度（HETP）、理论塔板数（N）和非对称因子（As），公式如下：
HETP = L / N
N = 5.54 × (Vʀ / Wₕ)²
As = a / b
其中：L为柱高； Vʀ为保留体积；Wₕ为半高峰宽；a为在10%峰高处的第一个半峰宽；b为在10%峰高处的第二个半峰宽。
一般来说，HETP的数值应小于填料平均粒径的三倍（即HETP/D50<3，D50为填料的平均粒径），As应在0.8~1.5之间。
3、平衡与上样
20 mM Tris-HCl, 500 mM NaCl, 1 mM MnCl₂, 1 mM CaCl₂, pH 7.4 是较为常用的结合缓冲液，也称为平衡缓冲液，记为缓冲液A。如果平衡时采用更高浓度的Mn²⁺和Ca²⁺，例如5 mM，可在结合时不添加Mn²⁺和Ca²⁺。结合缓冲液的pH在6.5~7.5之间为宜。
在上样前，需用缓冲液A在操作流速下平衡层析柱，该过程通常需要5~10个柱体积的缓冲液A，以电导、pH等检测信号参数不变且与缓冲液A对应为准。
上样量可按“mg目标蛋白/mL填料”来设定，通常为DBC10%的50~80%。在条件筛选阶段，也可以降低上样量，观察结合、特异性和洗脱情况。上样前需要对样品进行离心（10000 g 以上）、过滤（0.22或0.45 μm）处理，以免堵塞色谱柱。
上样后需要3~10个柱体积的缓冲液A再平衡层析柱。
4、洗脱
最常用的洗脱Buffer为0.1~0.5 M methyl-α-D-glucopyranoside（即 methyl-α-D glucoside、甲基 α-D-吡喃葡萄糖苷）或 methyl-α-D-mannopyranoside（即 methyl-α-D mannoside、甲基 α-D-吡喃甘露糖苷），20 mM Tris-HCl，500 mM NaCl，pH 7.4。可通过增加洗脱buffer中糖的浓度的方式洗脱蛋白，通常 0.1~0.2M 可洗脱大多数糖蛋白，但是更紧密结合的物质需要更高的浓度才能洗脱，在初次使用时尝试在5~500 mM范围内梯度洗脱。也可以通过降低pH来洗脱结合紧密的蛋白，但不能低于pH 4.0。也可以用较弱的洗脱剂如葡萄糖和甘露糖来洗脱。可以通过在洗脱过程中暂停流动几分钟来提高糖蛋白的回收率。
5、再生
在重复使用之前，可用5~10 CV的20 mM Tris-HCl，0.5 M NaCl，pH 8.5清洗，然 后用结合缓冲液平衡2~5 CV，平衡之后可进行下一次使用。也可在中间加入一步：20 mM NaAc，1 M NaCl，pH 4.5清洗2~5 CV。 对于吸附力强的物质可以用0.1 M硼酸盐缓冲液（pH 6.5~8.5），在低流速下清洗3~5 CV，硼酸可与糖基的邻二羟基形成复合物，达到竞争洗脱的目的。
6、填料保存
保存液为0.1 M acetate, 1 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂, pH 6.0，并使用20%乙醇防腐。保存温度在2~8℃为宜，不可冻存。
This product is a bioaffinity chromatography separation medium formed by covalently coupling Concanavalin A (Con A) to agarose gel microspheres. It is primarily used for the separation and purification of various glycoproteins (including glycoprotein viruses), polysaccharides, and glycolipids containing mannose and glucose residues. Con A, extracted from jack beans, is a tetrameric metalloprotein and a type of lectin. In the presence of Mn²⁺ and Ca²⁺, it binds to α-D-mannopyranosyl, α-D-glucopyranosyl, and molecules with spatially related C-3, C-4, or C-6 hydroxyl residues. Applications of Con A affinity chromatography medium also include purification of enzyme-antibody conjugates, IgM purification, isolation of detergent-solubilized cell surface glycoproteins from membranes, separation of membrane vesicles, and studies of changes in carbohydrate-containing substances.
Aladdin Con A Agarose Resin is stored in 20% (v/v) Ethanol, 0.1 M acetate, 1 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂, pH 6.0, 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
| Cross-linked Agarose
|
Ligand
| Con A, ≥8 mg/mL
|
Particle Size Range ①
| 45~165 μm
|
Average Particle Size
| ~90 μm
|
Binding Capacity ②
| ≥30 mg Thyroglobulin/mL
|
Recommended Operating Flow Rate ③
| 15~150 cm/h
|
Maximum Flow Rate & Pressure ④
| 900 cm/h, 0.3 MPa
|
Operating pH Range
| pH 4~9
|
Chemical Stability
| Stable in common aqueous buffers. Avoid chelators (e.g., EDTA), 8 M urea, or solutions below pH 3, as they may remove essential metal ions and inactivate the lectin.
|
Storage Conditions
| 20% (v/v) Ethanol, 0.1 M acetate, 1 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂, pH 6.0; 2–8°C
|
Shelf Life
| 3 years |
Notes:
① Over 90% of the beads fall within this size range.
② Binding capacity measured by incubating with 2 mg/mL porcine thyroglobulin for 2 hours; this value is close to the dynamic binding capacity (DBC₁₀%).
③ This flow rate range is recommended for optimal binding performance but is not an absolute operating limit.
④ Maximum tested flow rate at 10 cm column height.
Protocol
1. Column Packing
The following procedure describes column packing when connected to a chromatography system:
(1) Equilibrate all materials to the operating temperature. Degas liquids if possible.
(2) Resin Quantity Calculation:
Settled resin volume = Column volume × Compression factor (1.15 for this resin).
(3) Resin Preparation: Resuspend the resin slurry thoroughly. Measure the required volume, remove storage solution by filtration, and wash 3 times with ~3 resin volumes of purified water.
(4) Packing Slurry Preparation: Transfer resin to a suitable container and add packing solution to achieve a 50–75% (v/v) slurry. Mix well before use.
(5) Pre-packing Setup: Fill the bottom of the clean column with packing solution to remove air from the bottom frit and outlet. Retain a small amount of liquid, tighten the bottom end fitting, and ensure the column is vertical.
(6) Packing: Pour the well-mixed slurry into the column in one slow, continuous motion (use a packing reservoir if needed). After adding all resin, fill the reservoir with packing solution, attach the top lid, and connect the column to the system.
(7) Compression: Allow the resin to settle naturally (or use a low flow rate of 10–20 cm/h). Apply a high flow rate (300–600 cm/h; ensure pressure ≤0.3 MPa) until the interface is stable. Mark the stable height, stop the pump, and lower the adapter to the position corresponding to the compression factor (1.15). Tighten the adapter and equilibrate the column at a high flow rate.
Packing Condition
| Con A Agarose Resin
|
Compression Factor
| 1.15
|
Packing Flow Rate
| 300-600 cm/h |
2. Column Efficiency Testing
After packing, assess column efficiency using HETP (Height Equivalent to a Theoretical Plate) and As (Asymmetry Factor) with acetone or NaCl as tracers:
Tracer
| 1.0% Acetone
| 0.8~1.0 M NaCl
|
Sample Volume
| 1.0% CV
| 1.0% CV
|
Mobile Phase
| Pure Water
| 0.4 M NaCl
|
Flow Rate
| 30 cm/h
| 30 cm/h
|
Detector
| UV-280 nm
| Conductivity |
Calculate HETP, N, and As using:
HETP = L / N
N = 5.54 × (Vʀ / Wₕ)²
As = a / b
HETP should be <3× the average particle diameter (HETP/D₅₀ < 3), and As should be 0.8–1.5.
3. Equilibration and Loading
Recommended Binding Buffer (Buffer A): 20 mM Tris-HCl, 500 mM NaCl, 1 mM MnCl₂, 1 mM CaCl₂, pH 7.4. If higher concentrations of Mn²⁺ and Ca²⁺ (e.g., 5 mM) are used during equilibration, they may be omitted from the binding buffer. The pH of the binding buffer should be between 6.5–7.5.
Equilibrate the column with 5–10 CV of Buffer A at the operating flow rate until baseline (conductivity/pH) stabilizes.
Load sample at 50–80% of DBC₁₀%. For initial testing, reduce the load to observe binding, specificity, and elution. Pre-treat sample by centrifugation (≥10,000 g) and filtration (0.22/0.45 μm).
Wash with 3–10 CV of Buffer A after loading.
4. Elution
Common Elution Buffer: 0.1–0.5 M methyl-α-D-glucopyranoside (methyl-α-D-glucoside) or methyl-α-D-mannopyranoside (methyl-α-D-mannoside) in 20 mM Tris-HCl, 500 mM NaCl, pH 7.4.
Most glycoproteins elute at 0.1–0.2 M sugar concentration. Tightly bound substances may require higher concentrations. For initial use, try a gradient elution from 5–500 mM.
Alternatively, lower the pH (but not below 4.0) or use weaker eluents like glucose or mannose.
Tip: Pausing the flow for a few minutes during elution can improve glycoprotein recovery.
5. Regeneration
Before reuse, clean with 5–10 CV of 20 mM Tris-HCl, 0.5 M NaCl, pH 8.5, followed by equilibration with 2–5 CV of binding buffer.
Optionally, include a intermediate step: 2–5 CV of 20 mM NaAc, 1 M NaCl, pH 4.5.
For strongly adsorbed substances, use 0.1 M borate buffer (pH 6.5–8.5) at a low flow rate for 3–5 CV. Borate forms complexes with cis-diol groups of sugars, enabling competitive elution.
6. Storage
Store in 0.1 M acetate, 1 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂, pH 6.0, with 20% ethanol as preservative.
Store at 2–8°C. Do not freeze.
ZJ25F1129711
危险品化学品经营许可证（带存储）