SDS PAGE AND WESTERN BLOTTING
Western Blot Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.
Western Blot 聚丙烯酰氨凝胶电泳 SDS-PAGE 聚丙烯酰胺凝胶电泳 polyacrylamide gel electrophoresis 简称PAGE 作用 用于分离蛋白质和寡核苷酸 聚丙烯酰胺凝胶为网状结构 具有分子筛效应 它有 两种形式 非变性聚丙烯酰胺凝胶电泳 Native -PAGE 及SDS-聚丙烯酰胺凝胶 SDS-PAGE 非变性聚丙烯酰胺凝胶 在电泳的过程中 蛋白质能 够保持完整状态 并依据蛋白质的分子量大小 蛋白 质的形状及其所附带的电荷量而逐渐呈梯度分开 SDS-PAGE (十二烷基硫酸钠) 仅根据蛋白质亚基分 子量的不同就可以分开蛋白质
SDS 是阴离子去污剂, 作为变性剂和助溶试剂, 它能断裂分子内和分子间的氢键, 使分子去折叠, 破坏蛋白分子的二 三级结构 而强还原剂如巯基乙醇, 二硫苏糖醇 (DTT) 能使半胱氨酸残基间的二硫键断裂 在样品和凝胶中加入还原剂和 SDS 后, 分子被解聚成多肽链, 解聚后的氨基酸侧链和 SDS 结合成蛋白 - SDS 胶束, 所带的负电荷大大超过了蛋白原有的电荷量, 这样就消除了不同分子间的电荷差异和结构差异
考马斯亮蓝有 G-250 和 R-250 两种 考马斯亮蓝 G-250 在游离状态下呈红色, 最大光吸收在 465nm; 当它与蛋白质结合后变为青色, 蛋白质 - 色素结合物在 595nm 波长下有最大光吸收 其光吸收值与蛋白质含量成正比 G-250 由于与蛋白质的结合反应十分迅速, 常用来作为蛋白质含量的测定 R-250 与蛋白质反应虽然比较缓慢, 但是可以被洗脱下去, 所以可以用来对电泳条带染色
Prepare to transfer proteins to a nitrocellulose membrane ( 硝酸纤维膜 ) Trim gel
Mini Trans-Blot Transfer Cell
Preparing the Blotting Sandwich 1. Place the cassette with gray side down on clean surface 2. Place one pre-wetted fiber pad on the gray side of the cassette 3. Place a sheet of filter paper on the fiber pad 4. Place gel on filter paper taking care to remove air bubbles 5. Place the pre-wetted nitrocellulose membrane on the gel 6. Place the second fiber pad on top 7. Close the cassette firmly DO NOT move gel/filter sandwich 8. Lock the cassette
Prepare for Electrophoretic Transfer Place the closed and locked cassette in the electrode module Add the frozen Bio-Ice cooling unit and place in tank Fill the tank with buffer A stir bar can be added to help maintain the ion and temperature distribution in the tank even
Transfer Proteins from the gel to the nitrocellulose membrane 60 minutes 300mA Blotting buffer 1x Tris glycine with 20% methanol Electric Current
Blocking Buffer Remove membrane from the blotting sandwich and immerse in 25ml of blocking solution for 30 minutes 5% non-fat milk: Prevents the primary antibody from binding randomly to the membrane Phosphate buffered saline (PBS): Provides the correct environment (ph, Salt) to maintain protein shape 0.05% Tween 20: non-ionic detergent that prevents non-specific binding of antibodies to the membrane
Using the mammalian immune system to produce antibodies These antibodies are specific for our protein of interest
Use of antibodies as a diagnostic tool Molecule of interest is injected into primary animal model Animal makes antibodies against the molecule Antibodies are purified (primary antibody)
Use of antibodies as a diagnostic tool Antibodies from the first animal model are injected into a second animal model The second animal produces antibodies against the first antibody (secondary antibody) The secondary antibody is purified and conjugated to a colorimetric substrate or to an enzyme that can cleave a colorimetric compound
Add the Primary Antibody anti- pro- IL-1beta Discard blocking solution Pour 10ml (1:1000) of primary antibody onto the membrane and gently rock for overnight at 4 o C Primary antibody will bind to the pro-il1beta Wash Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer Add 50ml of wash leave for 3 minutes on the rocking platform
Add Enzyme-linked Secondary Antibody Discard wash solution Pour 10ml of the secondary antibody onto the membrane and gently rock for 60 minutes at room temperature Secondary antibody will bind to the primary antibody Wash Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer Add 50ml of wash leave for 3 minutes on the rocking platform Western Blot animation
Add Enzyme Substrate Discard wash solution Add 10ml of the enzyme substrate (HRP color detection reagent) onto the membrane Incubate for 10 minutes The colorimetric substrate is cleaved by the enzyme conjugated (attached) to the secondary antibody Watch for Color Development
Rinse and Store Rinse the developed membrane twice with distilled water and blot dry Air dry for 30min-1hr and store in lab notebook
LPS TLR4 K + NLRP3 ASC PYD PYD NACHT CARD LRR NF- B activation Nucleus ASC Deubiquitination NLRP3 NLRP3 IL1B Ubiquitin 1 Phosphorylation P Relocalization of NLRP3 Pro-caspase-1 2 Mitochondrion 3 ROS mtdna Cardiolipin K + Potassium efflux 4 Cathepsin release Lysosome Pro-caspase-1 NLRP3 Caspase-1 CARD p20/10 NLRP3 inflammasome ASC Pro-caspase-1 Debbie Maizels/Nature Publishing Group Haitao Guo et al. Nature Medicine 21, 677-687 (2015).