| Overview |
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| Ex/Em (nm) | None/None |
| MW | N/A |
| CAS # | N/A |
| Solvent | N/A |
| Storage | R |
| Category |
Protein Biochemistry General proteins |
| Related |
Biochemical Assays |
Upon receipt, store the kit at 4 oC. When stored properly, the kit should be stable for six months. Alternatively Components A and B can be stored at -20°C. Do not freeze Reaction Buffer (Component C) and Spin Column (Component D). Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following SOP is an example for labeling goat anti-mouse IgG antibody.
1. Prepare antibody solution :
For labeling 100 µg antibody (assuming the target antibodyconcentration is 1 mg/mL), mix 5 µL (5% of the total reaction volume) of Reaction Buffer (Component C) with 100 µL of the target antibody solution.
Note 1. If you have a difference concentration, adjust the antibody volume accordingly to make ~100 µg antibody available for your labeling reaction.
Note 2: The antibody should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4; If the antibody is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, or use Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa (cat# UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation.
Note 3: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.
Note 4: The antibody –Buccutite™ MTA reaction efficiency is significantly reduced if the antibody concentration is less than 1 mg/mL. For optimal labeling efficiency the final antibody concentration range of 1-10 mg/mL is recommended.
2. Run Antibody-Buccutite™ MTA reaction:
2.1 Add the antibody solution directly into the vial of Buccutite ™ MTA (Component B), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
2.2 Keep the antibody- Buccutite ™ MTA reaction mixture at room temperature for 30 - 60 minutes.
Note: The antibody-Buccutite™ MTA reaction mixture can be rotated or shaken for longer time if desired.
3. Prepare spin column for antibody-Buccutite™ MTA purification:
3.1 Invert the provided spin column (Component D) several times to re-suspend the settled gel and remove any bubbles.
3.2 Snap off the tip and place the column in a washing tube (2 mL, not provided). Remove the cap to allow the excess packing buffer to drain by gravity to the top of the gel bed. If column does not begin to flow, push cap back into column and remove it again to start the flow. Discard the drained buffer, and then place the column back into the Washing Tube. However, centrifuge immediately if the column is placed into a 12 x 75 mm test tube (not provided).
3.3 Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer.
3.4 Apply 1-2 mL 1X PBS (pH 7.2-7.4) to the column. After each application of PBS, let the buffer drain out by gravity, or centrifuge the column for 2 minutes to remove the buffer. Discard the buffer from the collection tube. Repeat this process for 3-4 times.
3.5 Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer.
4. Purify the antibody-Buccutite™ MTA solution:
4.1 Place the column (from Step 3.5) in a clean Collecting Tube (1.5 mL, not provided). Carefully load the sample (~105 μL, from Step 2.2) directly to the center of the column.
4.2 After loading the sample, add 5 μL of 1X PBS (pH 7.2-7.4) to make the total volume of 110 μL. Centrifuge the column for 5-6 minutes at 1,000 x g, and collect the solution that contains the desired antibody-Buccutite™ MTA solution.
5. Make HRP-antibody conjugation:
5.1 Make HRP- Buccutite™ FOL solution by adding 50 μL ddH2O into the vial of HRP- Buccutite™ FOL (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
5.2 Mix whole vial of HRP- Buccutite™ FOL solution (from Step 5.1) into the purified antibody- Buccutite™ MTA solution (from Step 4.2), mix well and rotating the mixture for 1 hour at room temperature.
5.3 The HRP-antibody conjugate is now ready to use.
Note 1: For immediate use, the HRP-antibody conjugate need be diluted with the buffer of your choice.
Note 2: For longer term storage, HRP-antibody conjugate solution need be concentrated or freeze dried.
| References & Citations |
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1. Presentini R, Terrana B. (1995) Influence of the antibody-peroxidase coupling methods on the conjugate stability and on the methodologies for the preservation of the activity in time. J Immunoassay, 16, 309.
2. Tresca JP, Ricoux R, Pontet M, Engler R. (1995) Comparative activity of peroxidase-antibody conjugates with periodate and glutaraldehyde coupling according to an enzyme immunoassay. Ann Biol Clin (Paris), 53, 227.
3. Strakova Z, Barancik M, Lukacova D, Angyal R, Slosarcikova L, Horakova K. (1991) Peroxidase labelled monoclonal antibody against light chains of human cardiac myosin. Gen Physiol Biophys, 10, 63.
4. Tijssen P, Kurstak E. (1984) Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays. Anal Biochem, 136, 451.
5. Boorsma DM, Cuello AC, van Leeuwen FW. (1982) Direct immunocytochemistry with a horseradish peroxidase-conjugated monoclonal antibody against substance P. J Histochem Cytochem, 30, 1211.
6. Tougard C, Tixier-Vidal A, Avrameas S. (1979) Comparison between peroxidase-conjugated antigen or antibody and peroxidase-anti-peroxidase complex in a postembedding procedure. J Histochem Cytochem, 27, 1630.
7. Sternberger LA, Petrali JP. (1977) The unlabeled antibody enzyme method. Attempted use of peroxidase-conjugated antigen as the third layer in the technique. J Histochem Cytochem, 25, 1036.
8. Broorsma DM, Steefkerk JG, Kors N. (1976) Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate. J Histochem Cytochem, 24, 1017.
AAT Bioquest AAT Bioquest是一家位于美国的生物公司，前身为ABD Bioquest，总部位于加利福尼亚州。专门从事光学检测技术十多年，一直致力于光谱学检测领域技术的创新和突破。其独特的光学检测技术，综合了化学、生物学和信息学等各个领域的研究，引领了比色、荧光和发光技术新一代光学探针的浪潮。AAT Bioquest在全球拥有强大的经验丰富的专业分销商网络，为从小型研究机构到《财富》500强企业的各类客户提供卓越的产品和定制服务。
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抗体都是免疫球蛋白而免疫球蛋白不一定都是抗体。原因是抗体是由浆细胞产生，且能与相应抗原特异性结合发挥免疫功能的球蛋白。而免疫球蛋白是具有抗体活性或化学结构与抗体相似的球蛋白，如骨髓瘤患者血清中异常增高的骨髓瘤蛋白，是由浆细胞瘤产生，其结构与抗体相似，但无免疫功能。因此，免疫球蛋白可看做是化学结构上的概念，抗体则是生物学功能上的概念。
二、保持乐观情绪乐观的态度可以维持人体于一个最佳的状态，尤其是在现今社会，人们面临的压力很大，巨大的心理压力会导致对人体免疫系统有抑制作用的荷尔蒙成分增多，所以容易受到感冒或其它疾病的侵袭。
三、限制饮酒每天饮低度白酒不要超过100毫升，黄酒不要超过250毫升，啤酒不要超过1瓶，因为酒精对人体的每一部分都会产生消极影响。即使喝葡萄酒可以降低胆固醇，也应该限制每天一杯，过量饮用会给血液与心脏等器官造成很大破坏。
四、参加运动专家进行的3项研究指出，每天运动30到45分钟，每周5天，持续12周后，免疫细胞数目会增加，抵抗力也相对增加。运动只要心跳加速即可，晚餐后散步就很适合。
五、补充维生素每天适当补充维生素和矿物质。专家指出，身体抵抗外来侵害的武器，包括干扰素及各类免疫细胞的数量与活力都和维生素与矿物质有关。
六、改善体内生态环境用微生态制剂提高免疫力的研究和使用由来已久。研究表明，以肠道双歧杆菌、乳酸杆菌为代表的有益菌群具有广谱的免疫原性，能刺激负责人体免疫的淋巴细胞分裂繁殖，同时还能调动非特异性免疫系统，去“吃”掉包括病毒、细菌、衣原体等在内的各种可致病的外来微生物，产生多种抗体，提高人体免疫能力。对于健康人来说，不妨“食疗”，多吃些乳酸菌饮料；而健康边缘人群，可以用微生态制剂来调节体内微生态平衡。能提高免疫力的食品
1.灵芝：灵芝可增强人体的免疫力，这是因为灵芝含有抗癌效能的多糖体，此外，还含有丰富的锗元素。锗能加速身体的新陈代谢，延缓细胞的衰老，能通过诱导人体产生干扰素而发挥其抗癌作用；
2.新鲜萝卜：因其含有丰富的干扰素诱导剂而具有免疫作用；
3.人参蜂王浆：能提高机体免疫力及内分泌的调节能力，并含具有防癌作用的蜂乳酸；
4.蘑菇、猴头菇、草菇、黑木耳、银耳、车养、百合等：都有明显增强免疫力的作用；
5.香菇：香菇所含的香菇多糖能增强人体免疫力。展开
