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变性/非变性细胞裂解产物制备试剂盒
描述：
非变性细胞裂解缓冲液内含非离子型去垢剂和盐，能裂解细胞并在非变性条件下释放胞浆蛋白和可溶性膜蛋白、核蛋白。非变性条件下的裂解蛋白产物最大限度地保留了蛋白的特性和功能，如抗原-抗体结合或酶学活性，因此适宜于进行免疫共沉淀。另外，有些抗体对非变性蛋白具有更高的结合能力或只能识别非变性蛋白的抗原位点，这种情况应该使用非变性裂解。
变性细胞裂解缓冲液用于变性裂解，适宜于如下几种情况：(1) 抗原是不溶性蛋白，如不溶性的细胞骨架和膜蛋白或高度疏水蛋白；(2) 抗原位点隐匿于蛋白高级折叠结构之中；(3) 易集聚沉淀的蛋白；(4) 体外翻译产生的蛋白。
在含离子型去垢剂的变性细胞裂解缓冲液中加热裂解细胞，可在变性条件下释放蛋白。随后，将变性蛋白裂解产物10倍稀释于非变性细胞裂解缓冲液中；变性裂解产物中的离子型去垢剂将被溶液中过量的非离子型去垢剂包裹进入到微小的微粒之中，从而恢复变性蛋白的抗原抗体结合活性。适宜于蛋白免疫共沉淀或Western Blot研究。
组成:
(100 preparations each for 0.5-2 x 107 cells )
(1) 非变性细胞裂解缓冲液100 ml;
(2) 变性细胞裂解缓冲液15 ml
适用：
蛋白免疫共沉淀，Western Blot
储存：
4 ºC避光。
制备非变性裂解产物
1. Collect cells: The number of cells necessary to detect an immunoprecipitated antigen depends on the cellular abundance of the antigen, and also on the efficiency of radiolabeling if performed. Approximately 0.5-2×10 7cells are required to yield 1 ml lysate, which is the amount generally used for each immunoprecipitation.
For adhesive cells growing at 80% to 90% confluence in a culture plate or flask:
Discard culture medium. Rinse cells twice with PBS. Place the tissue culture plate on ice. Add 1-1.5 ml (or suitable volume) ice-cold Nondenaturing Lysis Buffer to the culture plate, scrape the cells off the plate with a rubber policeman, and transfer the suspension to a 1.5-ml microcentrifuge tube. Go to Step 3.
Alternatively, adhesive cells can be disassociated with Non-enzyme Cell Detach Solution, which avoid using protease that degrades proteins. Collect cells as described below for nonadherent cells.
For nonadherent cells: Collect cells in suspension by centrifuging 5 min at 500× g at
4 oC. Discard supernatant. Wash cells twice with ice-cold PBS, and centrifuge as above.
2. Lysis of Cells. Add 1 ml (or suitable volume) ice-cold Nondenaturing Lysis Buffer to the cell pellet.
3. Extraction of cellular proteins: Vortex for 10 seconds. Keep suspension on ice for 20 min.
4. Centrifuge the lysate at 12,000 × g for 15 min, at 4 oC. The supernatant is transferred to a fresh tube, and now suitable in use for either immunoprecipitation or Western Blotting.
制备变性裂解产物
1. Collect cells in a 1.5 ml tube as described above (Step 1 on page 1). Place tube on ice.
2. Add 100 µl Denaturing Lysis Buffer per 0.5-2 × 10 7 cells in the pellet. Vortex for 10 seconds. The suspension may become viscous due to release of nuclear DNA.
3. Heat the sample at 95 oC for 5 min. Cool the sample on ice for 5 min.
（1）For Western Blot: Mixed the suspension with equal volume of standard SDS-sample buffer, the sample can be loaded directly on a SDS-PAGE gel. If the mixed lysate looks cloudy, centrifuge the sample as described in Step 4.
（2）For immunoprecipitation: Mix the suspension with 0.9 ml Nondenaturing Lysis Buffer 。Shear DNA by passing the suspension for 5~10 times through a 25-G needle attached to a 1-ml syringe. This step sequesters the ionic detergent into the micelles of nonionic surfactant, thus restore the antigen-antibody interaction.
4. Centrifuge the lysate at 12,000 × g for 10 min, at 4 oC. The supernatant is transferred to a fresh tube, and now suitable in use for either immunoprecipitation. Note that this preparation is also suitable in use for Western Blot.
NOTE:
1. Do not disturb the pellet by leaving the last 20 to 40 µl of supernatant in the centrifuge tube in step 4. Resuspension of even a small amount of pelleted material will result in high nonspecific background due to carryover into the immunoprecipitation steps. A cloudy layer of lipids floating on top of the supernatant usually does not affect the results of the immunoprecipitation.
2. Cell extracts can be frozen at -70 oC prior to use. However, it is preferable to lyse the cells immediately before immunoprecipitation in order to avoid protein degradation or dissociation of protein complexes. Some proteins can be actually degraded at least in part if the sample is stored under -20 oC for a few months even in the presence of the protease inhibitors. Note that many phosphorylated proteins are rapidly dephosphorylated within in few minutes or hours.
3.Protease inhibitors are not provided in the lysis buffer.
4.Nondenaturing Lysis Buffer (Cat# C1050) can be used as wash buffer for immuonprecipitation. |
