Recombinant Human His6-Ubiquitin-AA Mutant Protein, CF Summary
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins.Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration.The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard.In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
UM-HAA
Formulation | Lyophilized from a solution in deionized water. |
Reconstitution | Reconstitute at 5 mg/mL in an aqueous solution. |
Shipping | The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. |
Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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Reconstitution Calculator
Background: Ubiquitin
Ubiquitin is a 76 amino acid (aa) protein that is ubiquitously expressed in all eukaryotic organisms. Ubiquitin is highly conserved with 96% aa sequence identity shared between human and yeast Ubiquitin, and 100% aa sequence identity shared between human and mouse Ubiquitin (1). In mammals, four Ubiquitin genes encode for two Ubiquitin-ribosomal fusion proteins and two poly-Ubiquitin proteins. Cleavage of the Ubiquitin precursors by deubiquitinating enzymes gives rise to identical Ubiquitin monomers each with a predicted molecular weight of 8.6 kDa. Conjugation of Ubiquitin to target proteins involves the formation of an isopeptide bond between the C-terminal glycine residue of Ubiquitin and a lysine residue in the target protein. This process of conjugation, referred to as ubiquitination or ubiquitylation, is a multi-step process that requires three enzymes: a Ubiquitin-activating (E1) enzyme, a Ubiquitin-conjugating (E2) enzyme, and a Ubiquitin ligase (E3). Ubiquitination is classically recognized as a mechanism to target proteins for degradation and as a result, Ubiquitin was originally named ATP-dependent Proteolysis Factor 1 (APF-1) (2,3). In addition to protein degradation, ubiquitination has been shown to mediate a variety of biological processes such as signal transduction, endocytosis, and post-endocytic sorting (4-7).
Mature forms of Ubiquitin have a highly conserved diglycine motif at the carboxyl terminus which is crucial for activity and recognition in conjugation and deconjugation reactions. The replacement of this diglycine peptide with two alanine residues results in an inactive Ubiquitin. This Ubiquitin cannot be activated by the Ubiquitin-activating (E1) enzyme in an ATP-dependent manner, is not capable of subsequent thioester interaction with Ubiquitin-conjugating (E2) enzymes and/or Ubiquitin ligases (E3), and is thus not capable of forming isopeptide bonds or Ubiquitin conjugates. It can be used as a negative control in conjugation reactions, or in binding studies with Ubiquitin-activating (E1) enzymes, Ubiquitin-conjugating (E2) enzymes, Ubiquitin Ligases (E3), and DUB enzymes or other proteins that interact with Ubiquitin via Ubiquitin associated domains (UBAs) or Ubiquitin-interacting motifs (UIMs). This protein contains an N-terminal His6-tag.
- Sharp, P.M. & W.-H. Li. (1987) Trends Ecol. Evol. 2:328.
- Ciechanover, A. et al. (1980 ) Proc. Natl. Acad. Sci. USA 77:1365.
- Hershko, A. et al. (1980) Proc. Natl. Acad. Sci. USA 77:1783.
- Greene, W. et al. (2012) PLoS Pathog. 8:e1002703.
- Tong, X. et al. (2012) J. Biol. Chem. 287:25280.
- Wei, W. et al. (2004) Nature 428:194.
- Wertz, I.E. et al. (2004) Nature 430:694.
- Chau V. et al. (1989) Science. 243:1576-1583.
- Hass A.L. and Rose I.A. (1982) J.Biol.Chem. 257:10329-10337.
- Hass A.L. et al. (1985) J.Biol.Chem. 260:4694-4703.
- Reyes-Turcu R.E., et al. (2006) Cell. 124:1197-1208.
- Sloper-Mould K.E. et al. (2001) J.Biol.Chem. 276:30483-30489.
- Thrower J.S. et al. (2000) EMBO J. 19:94-102.
- Wilkinson K.D. and Audya T.K. (1981) J.Biol.Chem. 256:9235-9241.
FAQs
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Reconstitution Buffers
Reconstitution Buffer 1 (PBS)
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1、毕氏酵母氯化锂转化法
(1)试剂1M LiCl(用去离子蒸馏水配制,滤膜过滤除菌;必要时用消毒去离子水稀释)50% PEG3350(Sigma P3640 用去离子蒸馏水配制,滤膜过滤除菌,用较紧的盖子的瓶子分装)2mg/ml salmon sperm DNA / TE(10mM Tris-Cl, pH8.0, 1.0mM EDTA)-20℃保存注:醋酸锂对毕氏酵母无效,仅氯化锂有效;PEG3350可屏蔽高浓度LiCl的毒害作用;
(2)感受态毕氏酵母的制备接种Pachia pastoris到50ml YPD培养基中,30℃摇菌过夜(约24~28h)培养到OD值为0.8~1.0(约108 Cells/ml);收获细胞,用25ml无菌水洗涤一次,室温下1500g离心10min;重悬细胞于1ml 100mM LiCl溶液中,将悬液转入1.5ml离心管;离心机最大速度离心15秒沉淀菌体,重悬菌体于400ul 100mM LiCl溶液中;按50ul/管分装,立即进行转化;注:不要将感受态酵母菌冰浴;
(3)毕氏酵母的转化煮沸1ml鲑鱼精DNA 5min,迅速冰浴以制备单链担体DNA;将感受态酵母菌离心,以Tips去除残余的LiCl溶液;对于每一个转化,按以下顺序加入:50% PEG3350 240ul1M LiCl 36ul2mg/ml 单链Salmon sperm DNA 25ul5~10ug/50ul H2O 质粒DNA 50ul剧烈旋涡混匀直至沉淀菌体完全分布均匀(约1min);30℃水浴孵育30min;42℃水浴热休克20~25min;6000~8000rpm离心收集酵母菌体;重悬酵母于1ml YPD培养基,30℃摇床孵育;1~4h后,取25~100ul菌液铺选择性培养基平板,于30℃培养2~3天鉴定;
2、毕氏酵母PEG1000转化法
(1)试剂缓冲液A:1.0M Sorbitol,10mM Bicine,pH8.35(sigma),3%(v/v)ethylene glycol缓冲液B:40%(w/v)PEG1000(sigma),0.2M Bicine,pH8.35缓冲液C:0.15M NaCl,10mM Bicine,pH8.35未污染的新鲜、试剂级DMSO,-70℃保存注:缓冲液A、B、C均用滤膜过滤,-20℃保存;将DNA直接加在冻结的酵母细胞上是本实验的关键之处(即使在冰上解冻的待转化细胞,其摄取外源DNA的能力也在解冻过程中迅速下降;如进行多样品的转化,建议按6样品/组进行);
(2)待转化毕氏酵母的制备接种环接种Pachia pastoris于YPD平板,30℃培养2d;挑取单克隆酵母菌株于10mlYPD培养基中,30℃振荡培养过夜;取步骤2中小量菌液接种到100mlYPD培养基中振荡培养,待其OD值从0.1升到0.5~0.8;室温下3000g离心收集酵母菌体,50ml缓冲液A洗涤一次;重悬菌体于4ml缓冲液A中,按0.2ml/管分装于1.5ml的离心管中,每管加入11ulDMSO,混合后迅速于液氮中冷冻,-70℃保存
(3)毕氏酵母的转化将约50ug线性化质粒DNA溶于20ul TE或水中,直接加于冻结的酵母细胞中;加入担体DNA(40ug变性超声线性化鲑鱼精DNA)以获得最大转化率;37℃水浴孵育5min,中间混合样品1~2次;取出离心管,加入1.5ml缓冲液B,彻底混匀;30℃水浴孵育1h;室温下2000g离心10min,去除上清液,菌体沉淀重悬于1.5ml缓冲液C中;离心样品,去除上清液,轻微操作将样品重悬于0.2ml缓冲液C中;将所有转化液铺于选择性平板,于30℃孵育3~4天后,鉴定;
3、毕氏酵母电转化法
(1)E.coli TOP10F’感受态细胞的制备取10ul TOP10F’菌液,接种于200ml LB液体培养基中活化培养,37℃,200 rpm,16~18小时。取100ul菌液接种于200ml液体LB培养基中。37℃,200 rpm,培养16~18小时。灭菌500 ml离心管,4℃,4000 rpm,20 min。得菌体沉淀。弃上清,菌体用10%甘油重悬并洗涤。重复洗涤3次。第三次离心后,弃绝大部分上清,留下约1ml 液体用于重悬菌体。从制得得感受态细胞中,取200ul于灭菌EP管中,加入连接反应产物5ul,混匀,不要产生气泡,在冰上放置5min。将混匀后得200ul菌液移入电击杯中。使用电击穿孔仪进行转化,设置为电压 2500 V,时间 5 ms。电击后,往电击杯中加入 800ul SOC培养基,冲洗出菌体,转移至灭菌1.5 ml EP管中。37℃,150 rpm ,轻摇45~60 min。取全部均匀涂布于含 Zeocin 25 ug/ml 的LLB-Zeocin平板上,正放,待涂布液不在流动,37℃ 培养12~16小时。
(2)线性化质粒DNA对Pichia pastorisGS115的转化取新鲜制备的(或-70℃冻存的)感受态细胞置于冰浴中,使其完全解冻;
1)将100μl菌体移出至一新的无菌Eppendorf管中,加入5-20μg线性化质粒(5~10μl),轻弹混匀,尽数吸出转移到0.2cm型的电穿孔转化杯中;
2) 转化杯置于冰浴中5~10分钟,保持低温。
3) 电穿孔转化电击条件:电压:1500V;电阻:400Ω;电容:25μF;脉冲时间:10mS;一次电击。
4) 电击后,马上在电击转化杯中加入1ml 4℃预冷的1M的山梨醇溶液,用微量移液枪吹打均匀,置于冰浴中;5) 取30℃烘至表面半干的MD培养基平板,在超净工作台上无菌操作涂布平板,400μl/板
培养基:1640+10%FBS
消化条件: 预冷PBS浸泡5-10Min
细胞正常形态为圆形,贴壁生长,生长速度较快,细胞难消化,消化时用预冷的PBS淋洗,然后浸泡5min,让后用枪轻轻吹起吹散收集离心,这个过程很关键,如果没有消化到位就吹打或消化过久,细胞次日贴壁就会分化,分化之后的形态是细胞两端长出尖尖的触角,细胞不再圆形贴壁,而是平铺向四周扩散生长。
所以你换培养基试试看,我养的挺好的!
1. 配制含10%DMSO或甘油、10~20%小牛血清的冻存培养液;
2. 取对数生长期的细胞,去除旧培养液,用PBS清洗。
3. 去除PBS,加入适量胰蛋白酶(覆盖培养皿表面)把单层生长的细胞消化下来;
4. 离心1000rpm,5min;
5. 去除胰蛋白酶,加入适量配制好的冻存培养液,用吸管轻轻吹打使细胞均匀,计数,调节冻存液中细胞的最终密度为5×106/ml~1×107/ml;
6. 将细胞分装入冻存管中,每管1~1.5 ml;
7. 在冻存管上标明细胞的名称,冻存时间及操作者;
8. 冻存:标准的冻存程序为降温速率-1~-2℃/ min;当温度达-25℃以下时,可增至-5℃~-10℃/min;到-100℃时,则可迅速浸入液氮中。也可将装有细胞的冻存管放入-20℃冰箱2h ,然后放入-70℃冰箱中过夜,取出冻存管,移入液氮容器内。
吹打最好还是用弯头的,用起来更为方便
2×miRcute。
1. 37℃水浴预热培养液(HUVEC-004)。
2. 从液氮中取出HUVEC的细胞管,快速将其置入37℃水浴中解冻,直到管中只剩下最后一片结晶(注意:不要让水没过产品管)。 用75%的酒精擦拭产品管外壁。
3. 将HUVEC的细胞管中的细胞移至15ml无菌离心管中,慢慢逐滴加入预温的37℃ 基础培养液(HUVEC-004B)培养液4-5ml混匀,边加边摇匀。
4. 用1ml的培养液冲洗HUVEC的细胞管,向15ml离心管中加入冲洗的细胞悬液,边滴加边摇匀。
5. 取10μl细胞悬液和10μl台盼蓝混匀后,取10μl计数。
这一步骤非常重要,能确定您收到的产品是否合格(详见如何计数复苏的细胞)
6. HUVEC的细胞悬液在1500rpm,室温条件下,离心10分钟,去除上清。
7. 加HUVEC完全培养液4-5ml, 轻轻混匀细胞,备用。
8. 接种前将 T25培养瓶中的包被液吸去,按2500-5000个细胞/cm2接种细胞。(1支冷冻的细胞可以种一瓶T25培养瓶)
9. 每个HUVEC的细胞培养瓶中加完全培养液(HUVEC-004)3-5ml, 后置于37℃、5%CO2 培养箱内培养。
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