Westernblot实验技术.doc

Westernblot试剂配制
1.30%丙烯酰胺：
29.2g丙烯酰胺
用温热（利于溶解双丙烯酰胺）的去离子水（超纯水）溶后定容到100ml，过滤后于棕色瓶中储存
0.8gN,N’-亚甲双丙烯酰胺
注意：丙烯酰胺和双丙烯酰胺在贮存过程中缓慢转变为丙烯酸和双丙烯酸，这一脱氨基反映是光催化或碱催化的，故应核算溶液的pH值不超过7.0。这一溶液置棕色瓶中贮存于4℃，每隔几个月须重新配制。
小心：丙烯酰胺和双丙烯酰胺具有很强的神经毒性并容易吸附于皮肤。
2.10%SDS：
称5gSDS，超纯水溶解后，定容到50ml。贮存液保存于室温。
3.10%AP（过硫酸铵）：
称1gAP，溶于8ml超纯水中，定容到10ml，小量分装（250ul/管）保存于-20℃，
过硫酸铵提供驱动丙烯酰胺和双丙烯酰胺聚合所必需的自由基，每次使用均要用新鲜的。
4.1.5MTris-HCl：
称18.2gTris（Mr=121.14）加50ml超纯水溶解后，用浓HCl调pH值8.8，定容到100ml。
5.0.5MTris-HCl：
称3gTris（Mr=121.14）加25ml超纯水溶解后，调pH值6.8，定容到50ml。
6.TEMED(N,N,N’,N’-四甲基乙二胺)：
TEMED通过催化过硫酸铵形成自由基而加速丙烯酰胺与双丙烯酰胺的聚合。
一旦加入TEMED，立即开始聚合，故应立即快速旋动混合物并进入下步操作。
7.10×Tris-甘氨酸电泳缓冲液【10×（25mMTris+0.25M+0.1%SDS）】pH8.3
30.3gTris15.15gTris
187.65gGlycine溶于超纯水中，定容到1000ml93.825gGlycine5×缓冲液
10gSDS5gSDS
8.2×SDS上样缓冲液：2×（50mMTrispH6.8+2%SDS+10%甘油+0.1%溴酚蓝+50mMβ-巯基乙醇）loadingbuffer

10ml0.5MTris-HCl(pH6.8)
2gSDS
10ml甘油 超纯水定容到50ml。
0.1g溴酚蓝
5mlβ-巯基乙醇

9.Tris-甘氨酸SDS聚丙烯酰胺凝胶电泳分离胶溶液
溶液成分总体积
5ml总体积
10ml总体积
15ml总体积
20ml总体积
25ml总体积
30ml6%水2.6ml5.3ml7.9ml10.6ml13.2ml15.9ml30%丙烯酰胺1.0ml2.0ml3.0ml4.0ml5.0ml6.0ml1.5MTris(pH8.8)1.3ml2.5ml3.8ml5.0ml6.3ml7.5ml10%SDS0.05ml0.1ml0.15ml0.2ml0.25ml0.3ml10%过硫酸胺0.05ml0.1ml0.15ml0.2ml0.25ml0.3mlTEMED0.004ml0.008ml0.012ml0.016ml0.02ml0.024ml8%水2.3ml4.6ml6.9ml9.3ml11.5ml13.9ml30%丙烯酰胺1.3ml2.7ml4.0ml5.3ml6.7ml8.0ml1.5MTris(pH8.8)1.3ml2.5ml3.8ml5.0ml6.3ml7.5ml10%SDS0.05ml0.1ml0.15ml0.2ml0.25ml0.3ml10%过硫酸胺0.05ml0.1ml0.15ml0.2ml0.25ml0.3mlTEMED0.003ml0.006ml0.009ml0.012ml0.015ml0.018ml10%水1.9ml4.0ml5.9ml7.9ml9.9ml11.9ml30%丙烯酰胺1.7ml3.3ml5.0ml6.7ml8.3ml10.0ml1.5MTris(pH8.8)1.3ml2.5ml3.8ml5.0ml6.3ml7.5ml10%SDS0.05ml0.1ml0.15ml0.2ml0.25ml0.3ml10%过硫酸胺0.05ml0.1ml0.15ml0.2ml0.25ml0.3mlTEMED0.002ml0.004ml0.006ml0.008ml0.01ml0.012ml12%水1.6ml3.3ml4.9ml6.6ml8.2ml9.9ml30%丙烯酰胺2.0ml4.0ml6.0ml8.0ml10.0ml12.0ml1.5MTris(pH8.8)1.3ml2.5ml3.8ml5.0ml6.3ml7.5ml10%SDS0.05ml0.1ml0.15ml0.2ml0.25ml0.3ml10%过硫酸胺0.05ml0.1ml0.15ml0.2ml0.25ml0.3mlTEMED0.002ml0.004ml0.006ml0.008ml0.01ml0.012ml15%水1.1ml2.3ml3.4ml4.6ml5.7ml6.9ml30%丙烯酰胺2.5ml5.0ml7.5ml10