生物素亲和层析介质

产品介绍

储存缓冲液：20% (v/v) Ethanol 本产品是一种将D-生物素键合在琼脂糖基球上形成的亲和层析分离介质。D-生物素与链霉亲和素之间的亲和力非常高，其解离常数在10−14 mol/L数量级，是自然界最强的非共价相互作用之一。本产品特异性结合链霉亲和素后非常稳定，只有在苛刻的条件下，例如2%SDS的变性条件才能将链霉亲和素解离。因此该产品适用于亲和素、链霉亲和素及其偶联物的固定化，固定化后的产品可应用于诊断检测、蛋白纯化等。另外一方面，一些链霉亲和素的突变体与D-生物素之间的亲和力大幅减弱，使得突变体与D-生物素的特异性结合是可逆的，因而可以用该产品来纯化链霉亲和素突变体。阿拉丁生物素亲和层析介质储存在20%乙醇中，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6%交联琼脂糖配基D-生物素，≥5 μmol/mL粒径范围 ①45~165 μm平均粒径~90 μm动态结合载量 ②~30 mg 链霉亲和素突变体/mL介质推荐工作流速60~150 cm/h最大流速与压力 ③900 cm/h，0.3 MPa使用pH3~12（推荐的工作pH），3~13（CIP清洗）化学稳定性在以下溶液中稳定：常用的水相缓冲液、0.1 mol/L NaOH、8 mol/L尿素、2%吐温20等。储存20%乙醇，2~8℃保质期3年注：① 90%体积以上的微球在此粒径范围；② DBC10%测试条件为：10%流穿，6 min停留时间；③ 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。生物素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50~150 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。装柱条件生物素亲和层析介质压缩比1.15装柱流速600 cm/h2、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/h30 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 / NN = 5.54 × (Vʀ / Wₕ)²As = 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、推荐缓冲液推荐用以下缓冲液结合链霉亲和素或者纯化链霉亲和素突变体。结合缓冲液：20 mM Tris-HCl，150 mM NaCl，1 mM EDTA，pH 8.0洗脱缓冲液：结合缓冲液 + 10~50 mM D-生物素 再生缓冲液：0.1 M NaOH4、样品纯化（1）平衡：再用结合缓冲液平衡5个柱体积，建议流速为100~150 cm/h。（2）上样：将准备好的样品上柱，建议流速为20~100 cm/h，可根据实际结合情况选择流速，能获得较好的效果。上柱的样品应尽量保持与结合缓冲液一致。通常可用透析、超滤、稀释等方法处理样品。并且上柱前应过0.45μm滤膜或高速离心去除不溶物。 （3）再平衡：上样后用结合缓冲液平衡5~10个柱体积，或平衡至基线，洗去杂质，推荐流速为100~150 cm/h。（4）洗脱：用洗脱缓冲液洗6~10个柱体积，建议流速为100~150 cm/h，根据需要置换缓冲液。 （5）NaOH再生：洗脱目的蛋白后的柱子用纯水清洗3-5个柱体积，并用0.1 M NaOH再生3-5个柱体积，再用纯水清洗至中性，用20%乙醇保存柱子，或用结合缓冲液平衡后进行下一次纯化，建议流速为100~150 cm/h。5、填料保存 填料的初始保存液为20%乙醇，使用过后可继续用20%乙醇保存。保存温度在2~8 ℃为宜，不可冻存。
This product is an affinity chromatography separation medium formed by coupling D-biotin to agarose base beads. The affinity between D-biotin and streptavidin is extremely high, with a dissociation constant on the order of 10⁻¹⁴ mol/L, making it one of the strongest non-covalent interactions in nature. The binding between this product and streptavidin is highly stable and specific, requiring harsh conditions (e.g., 2% SDS denaturing buffer) for dissociation. Therefore, it is suitable for the immobilization of avidin, streptavidin, and their conjugates, with applications in diagnostic assays and protein purification. Additionally, some streptavidin mutants exhibit significantly reduced affinity for D-biotin, enabling reversible binding. This product can also be used to purify such streptavidin mutants.Aladdin Biotin Agarose Resin is stored in 20% ethanol, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel. ParameterValue / DescriptionMatrix6% cross-linked agaroseLigandD-biotin, ≥5 μmol/mLParticle Size Range ①45~165 μmAverage Particle Size~90 μmDynamic Binding Capacity ②~30 mg streptavidin mutant/mL resinRecommended Operating Flow Rate60~150 cm/hMaximum Flow Rate & Pressure ③900 cm/h, 0.3 MPaOperating pH Range3–12 (recommended), 3–13 (CIP)Chemical StabilityStable in common aqueous buffers, 0.1 M NaOH, 8 M urea, 2% Tween 20, etc.Storage Conditions20% ethanol, 2–8°CShelf Life3 yearsNotes:① Over 90% of the beads fall within this size range.② DBC₁₀% measured under: 10% breakthrough, 6 min residence time.③ Maximum tested flow rate at 10 cm column height.Protocol1. Column PackingThe 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 50–150 cm/h). Apply a high flow rate (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 ConditionBiotin Agarose ResinCompression Factor1.15Packing Flow Rate600 cm/h 2. Column Efficiency TestingAfter packing, assess column efficiency using HETP (Height Equivalent to a Theoretical Plate) and As (Asymmetry Factor) with acetone or NaCl as tracers:  Tracer1.0% Acetone0.8~1.0 M NaClSample Volume1.0% CV1.0% CVMobile PhasePure Water0.4 M NaClFlow Rate30 cm/h30 cm/hDetectorUV-280 nmConductivityCalculate HETP, N, and As using:HETP = L / NN = 5.54 × (Vʀ / Wₕ)²As = a / bHETP should be <3× the average particle diameter (HETP/D₅₀ < 3), and As should be 0.8–1.5.3. Recommended BuffersBinding Buffer: 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0Elution Buffer: Binding Buffer + 10–50 mM D-biotinRegeneration Buffer: 0.1 M NaOH4. Sample Purification(1) Equilibration: Equilibrate with 5 CV Binding Buffer at 100–150 cm/h.(2) Loading: Load sample at 20–100 cm/h. Pre-treat sample (dialysis, ultrafiltration, or dilution) to match Binding Buffer. Filter through 0.45 μm or centrifuge to remove particulates.(3) Wash: Wash with 5–10 CV Binding Buffer at 100–150 cm/h until baseline is stable.(4) Elution: Elute with 6–10 CV Elution Buffer at 100–150 cm/h.(5) Regeneration: Wash with 3–5 CV water, regenerate with 3–5 CV 0.1 M NaOH, rinse to neutral pH, and store in 20% ethanol or re-equilibrate with Binding Buffer.5. StorageStore in 20% ethanol at 2–8°C. Do not freeze.