Western blot analysis of lysates from wild type (WT) and 293T cells transfected with GSK3B Protein,BCAT2-N Protein,BCAT2-C Protein, using DDDDK-Tag antibody (AE092) at 1:10000 dilution.Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.Lysates/proteins: 25μg per lane.Blocking buffer: 3% nonfat dry milk in TBST.Detection: ECL Basic Kit (RM00020).Exposure time: 10s.
Western blot analysis of lysates from wild type (WT),293F transfected with COPB2-N Protein,COPB2-C Protein,using DDDDK-Tag antibody (AE092) at 1:10000 dilution.Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.Lysates/proteins: 25μg per lane.Blocking buffer: 3% nonfat dry milk in TBST.Detection: ECL Basic Kit (RM00020).Exposure time: 30s.
Chromatin immunoprecipitation was performed with 21.8 μg of cross-linked chromatin from 293T cells transfected with a GATA3 expression vector containing a single C-terminal DDDDK-Tag using 5 μg of Rabbit anti DDDDK-Tag (AE092). DNA libraries were prepared using Scale ssDNA-seq Lib Prep Kit for Illumina V2 (RK20228). The ChIP sequencing results indicate the enrichment pattern of DDDDK-Tag across chromosome 2 (upper panel) and the genomic region encompassing SH3RF3, a representative gene enriched in DDDDK-Tag (lower panel).
Chromatin immunoprecipitation was performed with 21.8 μg of cross-linked chromatin from 293T cells transfected with a GATA3 expression vector containing a single C-terminal DDDDK-Tag using 5 μg of Rabbit anti DDDDK-Tag (AE092). DNA libraries were prepared using Scale ssDNA-seq Lib Prep Kit for Illumina V2 (RK20228). The ChIP sequencing results indicate the enrichment pattern of DDDDK-Tag in the representative genomic region surrounding SH3RF3 gene.
Confocal imaging of 293F cells transfected with GFP-DDDDK-Tag (N) and 293F cells transfected with GFP-DDDDK-Tag (C) cells using DDDDK-Tag Rabbit mAb (AE092, dilution 1:1600) 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). Objective: 100x.
Immunoprecipitation of DDDDK-Tag from 300 ug extracts of 293T cells transfected with a SERPINB1 expression vector containing a single N-terminal DDDDK-Tag was performed using 3 μg of DDDDK-Tag Rabbit mAb(AE092). Rabbit IgG isotype control (AC042) was used to precipitate the Control IgG sample. The IP sample was eluted with 1X reducing Laemmli Buffer. The Input lane represents 10% of the total input. Western blot analysis of immunoprecipitates was conducted using DDDDK-Tag Rabbit mAb (AE092) at a dilution of 1:2000.
Immunoprecipitation of DDDDK-Tag from 300 ug extracts of 293T cells transfected with a GSK3B expression vector containing a single C-terminal DDDDK-Tag was performed using 3 μg of DDDDK-Tag Rabbit mAb(AE092). Rabbit IgG isotype control (AC042) was used to precipitate the Control IgG sample. The IP sample was eluted with 1X reducing Laemmli Buffer. The Input lane represents 10% of the total input. Western blot analysis of immunoprecipitates was conducted using DDDDK-Tag Rabbit mAb (AE092) at a dilution of 1:2000.
Chromatin immunoprecipitation was performed with 20 μg of cross-linked chromatin from 293F cells (left) and 293F cells transfected with BATF3 (right), using 2 μg of DDDDK-Tag Rabbit mAb (AE092) and Rabbit IgG isotype control (AC042). The enrichment of immunoprecipitated DNA at different genomic loci was examined by quantitative PCR. The histogram compares the ratio of the immunoprecipitated DNA to the input at given loci.
Flow cytometry: 1X10^6 293T cells (negative control,left) and 293T (Transfection,right) cells were surface-stained with DDDDK-Tag Rabbit mAb (AE092, 2.5 μg/mL orange line) or Rabbit IgG isotype control (AC042,2 μg/mL,blue line), followed by Alexa Fluor® 488 conjugated goat anti-rabbit pAb staining. Non-fluorescently stained cells were used as blank control (red line).
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FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology, having the sequence motif DYKDDDDK. It has been used for studying proteins in living cells and for protein purification by affinity chromatography. It has been used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits, because its mild purification procedure tends not to disrupt such complexes. It has been used to obtain proteins of sufficient purity and quality to carry out 3D structure determination by x-ray crystallography.A FLAG-tag can be used in many different assays that require recognition by an antibody. If there is no antibody against a given protein, adding a FLAG-tag to a protein allows the protein to be studied with an antibody against the FLAG sequence. Examples are cellular localization studies by immunofluorescence or detection by SDS PAGE protein electrophoresis and Western blotting.
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。
