Q1：在大肠杆菌蛋白质表达系统中，可以从细胞裂解物的可溶性部分或不可溶性部分纯化蛋白质吗？
A1：我们可以从两个组分中纯化蛋白质。它将取决于客户的实验需求或。我们的科学家在蛋白质复性方面有很好的经验。选择特定的复性策略是基于蛋白质的序列及其结构特性。
Q2：如果使用亲和标签，是否可以在纯化后把它从蛋白质中切割出来？
A2：是的，我们使用可以在纯化后被切割下来并从纯化的蛋白质中去除的标签（例如，通过在基因合成过程中引入凝血酶或TEV蛋白酶切割序列-一些氨基酸可能在切割后留在蛋白质上）。
Q3: 固定化抗体后，第一次亲和纯化蛋白效果良好，但在第二轮纯化过程中蛋白未能与抗体结合，是什么原因？
A3: 低pH洗脱是亲和纯化最常用的洗脱方法，但它可能导致某些抗体变性，从而对随后的抗原结合产生负面影响。为了防止这种情况发生，在蛋白质纯化之后，立即用中和或结合缓冲液洗涤柱子，并将其储存在含有抗菌剂如叠氮钠的缓冲液中，温度为4℃。或者，使用近中性，高盐洗脱缓冲液，如Pierce Gentle Ag/Ab Elution Buffer。
Q4: 纯化具有半胱氨酸的抗体,应该用什么办法呢？
A4: Sulfolink偶联树脂（SulfoLink Coupling Resin）或超临界碘乙醛树脂（UltraLink Iodoacetyl Resin）将是不错的选择，它们利用相同的化学反应与还原的硫醇基团反应。
Q5: 生物素亲和纯化是如何实现的？
A5: 固定化亲和素、链霉亲和素或中性亲和素用于纯化生物素裂解分子。这些分子与生物素的高亲和力要求不可逆变性以释放生物素本身(包括在SDS-PAGE样品缓冲液或8M胍、pH 1.5中沸腾)。
Q6: 能提供一些关于蛋白纯化后蛋白再折叠的建议吗？
A6: 请阅读下面的建议：
*保持低蛋白浓度10-50μg/ml。
*二硫键有助于稳定天然蛋白质；添加氧化还原对，如GSH（还原型谷胱甘肽）。
*GSSG（氧化型谷胱甘肽），其比为10:1，GSH浓度为2-5mm。这种氧化还原对有助于在折叠过程中产生破坏和建立新的S-S键的氧化电位。
*通过稀释或透析缓慢去除变性剂；甘氨酸(50mM，pH 9.0，5mM EDTA)有助于溶解蛋白质。如果使用GuHCl（盐酸胍），加入2M尿素，因为尿素也有助于稳定蛋白质折叠。
*在非常低的浓度下添加洗涤剂，例如0.1-0.5%的NP40或0.005%（V/V）Tween 20。
*包括用于稳定蛋白质的共溶剂，如甘油（5-20%）或PEG 8000或葡萄糖或蔗糖（10%）。
*某些阴离子(例如，磷酸盐或硫酸盐)或阳离子(例如，MES或HEPES)也具有积极作用；包括盐并保持中性pH，例如100mM KCl，或150-500mM NaCl，2mM MgCl2。
*通过添加蛋白酶抑制剂，如0.5mM PMSF、0.005-2g/mL抑肽酶、2g/mL抑肽酶或2-5g/mL亮肽素来避免蛋白质降解。
*磷酸盐缓冲液在抗钙时透析将导致磷酸钙沉淀。如果随后的肠激酶消化需要CaCl2（10mM Tris pH 8，10mM CaCl2），记得在透析完成后加入CaCl2。
Q7: 使用相同的亲和柱可以执行多少次纯化？
A7: 这随着固定化蛋白质的稳定性和所用洗脱缓冲液的类型而变化，通常可以重复使用色谱柱至少10次而不会显著损失活性。
卡梅德生物科技（天津）有限公司真诚期待与您的合作！
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