MMP8 Rabbit Recombinant mAb
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使用信息
| 抗体应用 | WB ,ELISA | ||
|---|---|---|---|
| 稀释比例 |
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| 反应性 | Human Mouse Rat | ||
| MW (kDa) | 53kDa | ||
| 抗体类型 | Rabbit | ||
| 浓度 | 1mg/ml | ||
| 储存液配方 | 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. | ||
| 储存条件 (自收到货起) |
Store at –20°C. |
生物描述
| 特异性 | MMP8 Rabbit Recombinant mAb detects endogenous level of total MMP8. |
|---|---|
| 背景 | Matrix metalloproteinases (MMPs) comprise a large family of zinc-dependent endoproteinases, collectively capable of degrading all extracellular matrix (ECM) components. They are synthesized as zymogens with a signal peptide which leads them to the secretory pathway, and then, they are secreted from the cell or anchored to the plasma membrane thereby confining their catalytic activity to the extracellular space or to the cell surface. Based on their domain organization, MMPs can be classified in four different groups: archetypal MMPs, matrilysins, gelatinases and furin-activatable MMPs. The archetypal MMPs then have been subdived into three different subgroups: collagenases, stromelysins and other archetypal MMPs. MMP8 belongs to teh group of collagenases which include MMP-1 and MMP-13 besides MMP8. Collagenases are also able to proteolytically process other ECM proteins, as well as a number of bioactive molecules such as interleukin-8 (IL-8), pro-tumor necrosis factor (TNF)-α, protease-activated receptor-1, and several insulin-like growth factor binding proteins (IGFBPs). The cooperation between the catalytic and hemopexin domains is essential to carry out their collagenolytic activity. They each has preferential activity against one of the three major types of fibrillar collagen: MMP-1 is mainly active against type III collagen, MMP-8 targets type I collagen and MMP-13 preferentially cleaves type II collagen |
实验方法
| WB |
Western Blotting Sample preparation 1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane. Membrane Blocking and Antibody Incubations a. Blocking 1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST. b. Antibodies Incubation 1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection. Detection of Proteins 1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film. |
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