MBP Tag 抗体
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北京 101111
Quick Overview for MBP Tag 抗体 (ABIN5542227)
抗原
宿主
克隆类型
标记
应用范围
克隆位点
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特异性
- This antibody reacts with MBP.
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纯化方法
- Protein A agarose
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免疫原
- pMALC2-G/JM109
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亚型
- IgG3
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anti-MBP Tag antibody
WB 宿主: 兔 Polyclonal RB55937 unconjugated
anti-MBP Tag antibody适用: 标记 WB, ELISA, IP, ICC, IHC, FACS 宿主: 小鼠 Monoclonal 3G1A3 unconjugated
anti-MBP Tag antibodyWB, ELISA, IP 宿主: 兔 Polyclonal unconjugated
anti-MBP Tag antibodyWB 宿主: 小鼠 Monoclonal 8K2 unconjugated
anti-MBP Tag antibodyWB, ELISA 宿主: 小鼠 Monoclonal 5B10B11 unconjugated
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应用备注
- Western blot: 1 μg/mL. Immunoprecipitation: 2 μg/100 μg of E. coli lysate. For details see protocol below.
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实验流程
- SDS-PAGE & Western Blotting 1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the Lysis buffer to make an 8 mg/mL solution. 3) Mix the sample with an equal volume of Laemmli's sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 μ L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20 % MeOH). See the manufacture's manual for specific transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1 % skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The optimal antibody concentration will depend on the experimental conditions.) 8) Wash the membrane with PBS (5 minutes x 6 times). 9) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG diluted with 1 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS (5 minutes x 6 times). 11) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap. 12) Expose to X-ray film in a dark room for 5 minutes. Develop the film as usual. The optimal conditions for exposure and development may vary. Immunoprecipitation 1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. 3) Add primary antibody as suggested in the APPLICATIONS into 100 μ g of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4 o C. Add 20 μ L of 50 % protein A agarose beads resuspended in the cold Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4 o C. 4) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds). 5) Resuspend the beads in 20 μ L of Laemmli's sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10 μ L/lane for the SDS-PAGE analysis. (See SDS-PAGE & Western blotting)
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限制
- 仅限研究用
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状态
- Liquid
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缓冲液
- PBS containing 50 % glycerol, pH 7.2. Contains no preservatives.
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储存液
- Without preservative
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储存条件
- -20 °C
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储存方法
- Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing. Shelf life: One year from despatch.
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- MBP Tag
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别名
- maltose binding protein tag,mbp-tag
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物质类
- Tag
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背景
- Maltose Binding Protein (MBP) is a part of the maltose/maltodextrin system of Eschericia coli bacteria, which is responsible for the uptake and efficient catabolism of maltodextrins. It is a complex regulatory and transport system involving many proteins and protein complexes. MBP is often used as a fusion partner for recombinant protein expressi on, primarily because its affinity for maltodextrins allo ws for facile purification of fusion proteins by amylose affinity chromatography.
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UniProt
- P0AEX9
抗原
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