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Day 0 and 1 Materials 1.0.5 mM ATP0.2 mM CaCl21 mM PIPESpH 6.8 at 4℃250 ml for day 0. 2.0.5 mM ATP0.2 mM CaCl270 mM KCl150 mM PIPESpH 7.01 ml. 3.16 mM ATP0.2 mM CaCl25 mM DTT5 mM lysine10 mM glutamic ...

Day 0 and 1 Materials 1.0.5 mM ATP0.2 mM CaCl22 mM Tris-HClpH 8.0 at 4℃250 ml for day 0 and 1000 ml for day 1. 2.100 mM KCl100 mM boric acidpH 8.4 at 4℃10 ml. 3.IATR (tetramethylrhodamine iodoacetamid ...

1、侵袭实验与转染细胞问题。 问：我要做侵袭实验，但我做的是瞬时转染的贴壁细胞，我想请教各位，瞬时转染后多长时间，可以消化细胞进行transwell小室的细胞接种？ 答：最好是24小时，我们科有两个都用二十四小时，但也有文献报道说用48小时。如果是稳定转染，则需要在对数生长期接种细胞。 2、原代成骨细胞培养不贴壁问题。 问：我用新配置的0.25%胰蛋白酶消化骨片30分，然后用胶原酶消化90分，培养 ...

Day 0 and 1 Materials 1.0.5 mM ATP0.2 mM CaCl22 mM Tris-HClpH 8.0 at 4℃250 ml for day 0. 2.0.5 mM ATP2 mM MgCl2100 mM boric acidpH 8.3 at 4℃10 ml. 3.N-(1-pyrene)iodoacetamide (Molecular probes P-29m.w ...

Day 1-2 Materials 1.0.5 M KCl10 mM HEPESpH 7.54℃250 ml. 2.0.5 M KCl50 mM HEPESpH 8.04℃250 ml. 3.Bio-Beads SM-2 in a 0.7x15 column. 4.20 mM KCl20 mM PIPESpH 7.04℃250 ml. 5.IATR (tetramethylrhodamine io ...

Day 1 Materials 1.50 mM KCl0.2 mM EGTA2 mM ATP2 mM MgCl210 mM HEPESpH 7.0.Need 500 ml. Procedure 1.Thaw appropriate volume of frozen gizzard myosin to obtain 10-20 mg. 2.Centrifuge in a SS34 rotor at ...

Materials 1.Small vial and stir bar. 2.Bio-Beads SM-2 in 0.7x15 cm column. 3.2 mM Tris-HClpH 8.54℃250 ml. 4.Centricon-30 concentrator. 5.5 mM Tris-acetate0.1 mM DTTpH 6.954℃250 ml. 6.200 mM K-boratepH ...

Materials 1.200 mM K-boratepH 9.010 ml. 2.100 mM lysine100 mM K-boratepH 9.01 ml aliquots stored at -20℃. 3.2 mM Tris-HClpH 8.54℃250 ml. 4.100 mM DTT stock. 5.Centricon-30 concentrator. 6.5 mM Tris-ac ...

Materials 1.Drop frozen 2x cycled tubulin in a polymerization-promoting buffer.Stored at -80℃ 2.GTP100 mM stocktitrate to pH ~7.Need 2 ml. 3.Waterbathset at 37℃ 4.SS34 rotor5 ml and 15 ml tubes and ad ...

Materials 1.2 mM PIPESpH 7.0500 ml. 2.Gc-globulin (Calbiochem 345802)1 mg. 3.CFSE (Carboxyfluorescein succinimidyl ester; Molecular Probes). 4.Lysine100 mM in buffer 110 ml. 5.G25-150 desalting column ...

Considerations for use The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay.It is fairly accurate and samples that are out of range can be retested within minut ...

Considerations for use The principle of the biuret assay is similar to that of the Lowryhowever it involves a single incubation of 20 min.There are very few interfering agents (ammonium salts being on ...

Considerations for use The bicinchoninic acid (BCA)assay is available in kit form from Pierce (RockfordIll.).This procedure is very applicable to microtiter plate methods.The BCA is used for the same ...

How long should cells be labeled? The ideal length of time to label cells depends on the protein of interest and the label that you are using.If you want to label an unstable protein with 35S-methioni ...

Recipes for the buffers are at the end of this protocol. 1.Label your protein with 32Pi.Then subject the protein to SDS polyacrylamide gel electrophoresis and transfer your gel-fractionated protein to ...

This specific procedure was developed to assay the activity of the Lck kinase expressed from a retroviral vector in rat fibroblasts.You can obviously use fewer or more cellsdepending on your needs.Don ...

Preparation of protein extracts for western blot 1.Grow cells to mid-log (OD600 less or equal to 1.0). 2.Harvest about 5 OD's of cells in a 13X100 glass dispo tube (Use new tubes.Can be up to about 6 ...

问：我要检测痰中弹性蛋白酶的活力，买的是sigma的底物，在410nm处比色，请问最后酶活力用什么单位来表示呢？加完底物后，立即比色，我隔5分钟就测定一次，一共测了一个小时。最后只得出了OD值，看相关书籍及各位战友以前的帖子，发现需要划出标准曲线，请各位高手指教，具体的测酶活力的具体步骤好吗？要做哪些标准曲线呢？谢谢！ 答：您每5min进行一次测定，我想目的应该是对酶反应时间进行考察，从而得出弹性 ...

问：用胰蛋白酶切蛋白需要加氯化钙吗？急！ 答：是要活化， 37度4小时， 或者30度 overnight with shaking。 问：我还想问一下，用什么作为活化液？我用的是三乙醇胺行吗？ 答：氯化钙是用来配制酶解缓冲液的，一般为5毫摩每升，也可以用已睛代替.或者就直接用40毫摩每升的碳酸氢胺。 我不知道你买的酶是哪个公司的，如果是SIGMA的，就不需要活化了，可以直接按照其说明书上的说明 ...

问：蛋白酶解肽段的纯化，去除杂质，去除，即可，不需要分离！请问怎么设计色谱条件呢？柱子，填料，流动相，流速？？谢谢！！ 问题比较菜！ 答：要看你的蛋白和酶解以后片断的大小了，楼主是要保留蛋白片断去除酶解buffer 里的东西么？一般根据样品中蛋白的大小和疏水性选择不同的柱子。常用的是反相疏水柱，几千的蛋白可以用C18硅胶键合柱，上万的要用C3的了。流动相主要是乙氰和水，流速要根据你的柱子大小而定 ...