Western blot analysis of lysates from HeLa cells using 4-Hydroxynonenal Rabbit mAb (A26085) at 1:10000 dilution incubated overnight at 4℃. HeLa cells were treated with 4-HNE (0.2 mg/ml) at 37℃ for 30 minutes.Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.Lysates/proteins: 30 μg per lane. Blocking buffer: 3 % nonfat dry milk in TBST. Detection: ECL Basic Kit (RM00020). Exposure time: 30s.
Confocal imaging of HeLa cells (treated with 4-HNE) and HeLa cells (untreated) using 4-Hydroxynonenal Rabbit mAb (A26085, dilution 1:200) followed by a further incubation with Cy3 Goat Anti-Rabbit IgG (H+L) (AS007, dilution 1:500) (Red). The cells were counterstained with α-Tubulin Mouse mAb (AC012, dilution 1:400) followed by incubation with ABflo® 488-conjugated Goat Anti-Mouse IgG (H+L) Ab (AS076, dilution 1:500) (Green). DAPI was used for nuclear staining (Blue). Objective: 100x.
Confocal imaging of paraffin-embedded Mouse brain and 5xFAD Mouse brain tissue using 4-Hydroxynonenal Rabbit mAb (A26085, dilution 1:200) followed by a further incubation with Cy3 Goat Anti-Rabbit IgG (H+L) (AS007, dilution 1:500) (Red). DAPI was used for nuclear staining (Blue). High pressure antigen retrieval performed with 0.01M Citrate Buffer (pH 6.0) prior to IF staining. Objective: 40x.
Confocal imaging of HeLa (treated with 4-HNE) and HeLa(untreated) cells using 4-Hydroxynonenal Rabbit mAb (A26085, dilution 1:200) followed by a further incubation with Cy3 Goat Anti-Rabbit IgG (H+L) (AS007, dilution 1:500) (Red). The cells were counterstained with α-Tubulin Mouse mAb (AC012, dilution 1:400) followed by incubation with ABflo® 488-conjugated Goat Anti-Mouse IgG (H+L) Ab (AS076, dilution 1:500) (Green). DAPI was used for nuclear staining (Blue). Objective: 100x.
Flow cytometry: 1X10^6 HeLa cells (negative control,left) and HeLa cells (treated with 4-Hydroxynonenal,right) were intracellularly-stained with 4-Hydroxynonenal Rabbit mAb (A26085,2 μg/mL,orange line) or Rabbit IgG isotype control (AC042,2 μg/mL,blue line), followed by FITC conjugated goat anti-Rabbit pAb staining. Non-fluorescently stained cells were used as blank control (red line).
Flow cytometry: 1X10^6 HeLa cells (treated with 4-Hydroxynonenal) were intracellularly stained with Rabbit IgG isotype control (AC042,2 μg/mL,left) or 4-Hydroxynonenal Rabbit mAb (A26085,2 μg/mL,right), followed by FITC conjugated goat anti-Rabbit pAb staining.
| IF (Immunofluorescence) | Mouse |
|---|---|
| IHC (Immunohistochemistry) | Human |
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4-hydroxy-2-nonenal (4-hydroxynonenal, 4-HNE) is a highly reactive aldehyde generated by the exposure of polyunsaturated fatty acids to peroxides and reactive oxygen species (ROS). It non-enzymatically forms stable protein adducts with histidine, lysine, and cysteine side chains that have been used as biomarkers for oxidative damage in cells. Conditions where 4-HNE immunoreactivity has been observed include include inflammation, neurodegenerative diseases, and ischemic damage to the heart and brain.
首先，一般抗体不推荐客户回收利用，抗体使用之后缓冲体系已经发生改变，不同客户在回收抗体的保存条件上也会有差异，所以抗体回收使用效果无法保证。另外，ABclonal公司也做过一批抗体回收验证测试，测试结果显示不同抗体可回收次数不同，一般效价越高的抗体，可重复使用的次数越多，客户可根据实验情况来确定。
注：我们将孵育完毕后剩余的抗体回收到离心管中置于4℃保存，效价高的抗体可至少保存1周，至少重复利用3次。
武汉爱博泰克生物（ABclonal）科技有限公司是国产品牌，她成立于2011年，公司依托ABclonal美国波士顿抗体与蛋白研发中心、中国光谷生物城(武汉)抗体生产基地以及上海张江分子酶研发中心，凝聚了十余位来自哈佛大学、麻省理工、复旦大学、上海交大、中科院生化细胞所和武汉大学的全球知名分子和免疫学方面博士，组成我们的科学家团队，通过聚焦抗体与酶核心技术，致力于打破国际技术的垄断，将公司打造成为科研工具和诊断原料的国内领导品牌，乃至弯道超越国际巨头。 我们拥有包括兔多克隆抗体、小鼠单克隆抗体、兔单克隆抗体的生产研发平台，同时也有包括WB,IHC,IF,IP,CHIP在内的检测平台，我们对每一支自产的抗体进行了严格的检测。当然，我们部分直销地区也可以帮客户代购进口品牌的产品。同时也有抗体定制服务。ABclonal抗体优势：1，严自检，保质量；2产品多，指标全；3，价格低，货期短。注：ABclonal抗体价格体系详情见附件
ABclonal抗体成分在平时工作当中，常会有客户咨询我们的抗体用的什么buffer进行保存，一般来说，我们的buffer的成分是：PBS含0.03%的proclin300、0.05%牛血清白蛋白、50%甘油；也有一些是PBS含0.03%的proclin300，50%甘油。防腐剂 Proclin 300活性成分主要是2-甲基-4-异噻唑啉-3-酮（MCI）和5-氯-2-甲基-4-异噻唑啉-3-酮（CMCI）。ProClin生物灭活剂能够迅速穿透细胞膜，抑制对细胞呼吸至关重要的特定酶，因此一接触微生物有机体就会立即抑制细胞活性。ProClin的多个特定毒性位点可以防止微生物产生高水平的耐药性。
