pCold ProS2 DNA is a fusion cold-shock expression vector whichfeatures Protein S, a soluble tag from Myxococcus xanthus. Protein S, which is comprised of 173 amino acid residues, is a very stable soluble protein present in a spore surface coat of Myxococcus xanthus. A fusion of target protein with the ProS2 Tag, a tandem repeat of 2 N-terminal domain (NTD) sequences of Protein S, will induce the stability and solubility of the fusion protein.
pCold ProS2 DNA is a fusion cold-shock expression vector whichfeatures Protein S, a soluble tag from Myxococcus xanthus. Protein S, which is comprised of 173 amino acid residues, is a very stable soluble protein present in a spore surface coat of Myxococcus xanthus. A fusion of target protein with the ProS2 Tag, a tandem repeat of 2 N-terminal domain (NTD) sequences of Protein S, will induce the stability and solubility of the fusion protein.
The pCold ProS2 DNA Vector consists of the cspA promoter plus additional downstream sequences including a 5" untranslated region (5" UTR), translation enhancing element (TEE), his-tag sequence, ProS2 tag, and multiple cloning site (MCS). A lac operator is inserted downstream of the cspA promoter to ensure strict regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin, and Factor Xa are located between the ProS2 Tag and MCS and function to facilitate tag removal from the expressed fusion protein. Most E. coli strains can serve as expression hosts. The pCold ProS2 DNA vector provides cold-shock technology for high yield protein expression to facilitate correct protein folding, thus enabling efficient soluble protein production for otherwise intractable target proteins.Elucidation of protein structure and function maintains an important role in postgenomic sequencing and analysis studies. An efficient protein production system is critical for obtaining large amounts of correctly folded recombinant protein for study. E. coli expression systems, which are used extensively for the production of recombinant proteins, offer two major advantages over other types of expression systems: 1) ease of use, and 2) low cost. However, some recombinant proteins do not fold correctly during expression in E. coli and result in deposits of inactive insoluble protein termed "inclusion bodies."Takara Bio has developed the pCold DNA vectors, a series of novel protein expression vectors in collaboration with Prof. Masayori Inouye (University of Medicine and Dentistry of New Jersey, USA). The pCold Vectors provide increased in vivo protein yield, purity, and solubility for expressed recombinant proteins using "cold shock" technology. More specifically, the cspA (cold shock protein A) promoter and related elements have been incorporated into these vectors to up-regulate target protein production at lowered incubation temperatures (37–15°C). This temperature drop also suppresses expression of other cellular proteins and temporarily halts overall cell growth. This process allows high-yield, high-purity expression of target proteins (up to 60% of cellular protein), and increased solubility as compared with conventional E. coli expression systems.
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望不吝赐教！
但是His标签是目前最普及最方便的融合标签，所以His标签的抗体，不管是单抗还是多抗，鼠源还是兔源都是商品化程度非常高的的抗体。这样的话因为抗体原因失败的可能性就非常小了。
单抗标签选择的一抗范围比较小一些，多抗标签选择的一抗范围可能相对大一些。而商品化二抗基本没什么差别。不管是选择什么样的抗体，在Western blot检测方面都是一样的
所以做Western blot的单抗和多抗标签并没有什么不同
（2）6 X His tag 序列：CAT CAT CAC CAT CAC CAT
（3）GST标签序列：
1ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGC
301 AACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCAT
351 TTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATT
401 GGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAAT
451 TAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATG
501 TTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGC
551 GGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACT
601 TTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAA
651 ATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGT
701 AACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACA
751 TGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAA
801 CGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTA
851 TATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACC
901 ATCCTCCAAAATCGGATCTGGTTCCGCGTGGATCCCCGGAATTCCCGGGT
951 CGACTCGAGCGGCCGCATCGTGACTGA
百度上的，，，，，搬运
求助：为什么用蛋白抗体仅出来一条带，而用Flag抗体则出来两条带？
谢谢大家啊
图一：蛋白抗体
图二Flag抗体
Flag标签系统是已被公认的用于表达、纯化以及检测融合蛋白的表达系统，广泛应用于Westernblotting、免疫细胞组化、免疫共沉淀、流式细胞术、蛋白纯化以及蛋白相互作用、蛋白分离等多个研究领域。Flag标签是一个与重组蛋白相融合的由8个亲水氨基酸（Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys）组成的多肽片断。FLAG标签只有8个氨基酸组成，因此它不会占据其他表位与结合域，避免导致融合蛋白的功能、分泌性以及转运发生改变。由于它的亲水性特点，FLAG标签倾向于定位在融合蛋白表面，因此更易与抗体结合以及被肠激酶酶解。
由于Flag标签可位于蛋白质的C端或N端，这一系统已用于各种细胞类型，包括细菌、酵母和哺乳细胞。由于Flag标签系统的纯化条件是非变性的，因此可以纯化所有有活性的融合蛋白。EarthOx的Anti-FlagTagMonoclonalAntibody可以用于检测和Flag标签融合表达蛋白的表达、细胞内定位，以及纯化、定性或定量检测Flag融合表达蛋白等。比如在免疫荧光IF的实验里，同时配合EarthOx的Dylight549荧光二抗，使用该抗体可以准确的定位出Flag融合表达蛋白在细胞内的位置（如图红色显示部分）。另外，做融合表达分析前，通过免疫共沉淀技术获得Flag融合表达蛋白也是非常重要的一步，因此选择一个合适的、可用于免疫共沉淀（Immunoprecipitation，IP）Flag标签抗体也是很必要的。EarthOx的Anti-FlagTagMonoclonalAntibody不仅能应用于细胞内定位的IF实验，同样还能应用于IP实验。1:200甚至更高的IP实验稀释比率，也说明了该抗体本身的具有很高的效价，同时，因为是单克隆抗体，因此特异性也很高。高效价所对应的高稀释比率也意味着超高性价比。
