Product Specifications:
Item# 1008: Recombinant HIV-1 nef (E.coli)
Concentration: See vial
Mass/vial: 100 ug
Volume/vial: 100ul
Diluent: 50mM Tris, pH8, 150mM NaCl
Purity: >95%
Stabilizer: None
Preservative: None
Storage: -75°C
Physical State: Frozen Liquid
Stability: Minimum 12 months at -75°C.
Application: Diagnostics, ELISA/ Western ELISA, Drug Screening, Immunological Studies.
Description: Full length Recombinant HIV-1 (LAV) nef produced as a GST fusion protein in E.coli.
Purification: This recombinant protein is purified by Glutathion -affinity chromatography and the purified protein is thrombin-cleaved to produce nef of >95% purity, as determined by SDS-PAGE, reduced.
Specificity: This protein shows reactivity with murine monoclonal antibodies and human polyclonal antibodies in ELISA and Western ELISA.
Biological Activity: Not done.
Application and Instructions for use
Recommended concentrations for use are approximate values. A dose dependent response assay should be performed to determine the optimal concentration for use in specific applications. ELISA and Western ELISA require 1-10ng protein depending on the nature and affinity of the test antibody.
Glossary
Gene and Gene Products
Structural Proteins: Structural proteins – the products of gag, pol and env genes, which are essential components of the retroviral particle.
Regulatory Proteins: Regulatory proteins – tat and rev proteins of HIV/SIV and tax and rex proteins of HTLVs; essential for viral expression in infected cells.
Accessory Proteins: Accessory proteins – additional (non-regulatory) virion – and non virion-associated proteins produced by HIV/SIV retroviruses: vif, vpr, vpu, vpx, and nef. Although, the accessory proteins are not necessary for viral propagation in tissue culture, they have been conserved in the different isolates; this conservation and experimental observations suggest that their role in vivo is very important.
gag
gag – group-sepecifc antigens or capsid proteins; the precursor is the p55 myristoylated protein, which is processed to p17 (Matrix) p24 (Capsid) and p7 (NucleoCapsid) proteins by the viral protease. Other small proteins are generated from the gag polyprotein.
pol
pol – (p66) generates the viral enzymes protease (p11), reverse transcriptase (p51), endonuclease and integrase (p32) after the processing of a gag-pol precursor polyprotein by the viral protease; gag-pol precursor is produced by ribosome frameshifting.
env
env – viral glycoproteins produced as a precursor (gp160) and processed to the external glycoprotein (gp120) and the transmembrane glycoprotein (gp41). The mature proteins are held together by noncovalent interactions; as a result substantial amount of gp120 is released extracellularly. The external glycoprotein (gp120) contains the binding site for the CD4 receptor.
tat
tat – transactivator of HIV gene expression; one of the two necessary viral regulatory factors (tat and rev) for HIV gene expression. Two forms are known, tat-1 exon (minor form) of 72 amino acids, and tat-2 exon (major form) of 86 amino acids. The electrophoretic mobility of these two forms in SDS gels is anomalous; they are approximately 16 kD and 14 kD in weight. Low levels of both proteins are found in persistently infected cells. tat is localized primarily in the nucleolus/nucleus; it acts by binding to the TAR RNA element and activating transcription from the LTR promoter. Post-transcriptional effects of tat have been postulated.
rev
rev – the second necessary regulatory factor for HIV expression. A 19 kD phosphoprotein localized primarily in the nucleolus/nucleus, rev acts by binding to RRE and promoting the nuclear export, stabilization and utilization of the viral mRNAs containing RRE.
vif
vif – viral infectivity factor, typically 23 kD; required for the efficient transmission of cell-free virus in tissue culture. In the absence of vif, the produced viral particles are defective, while the cell-to-cell transmission of virus is not affected significantly. It has been reported that the cellular localization is in the Golgi (vif is not found in the virion).
nef
nef – approximately 27 kD non-virion protein found in the cytoplasm of infected cells. Potentially myristoylated and associated with the inner plasma membrane. One of the first HIV proteins to be produced in the infected cells, it is the most immunogenic of the accessory proteins and may be used in the future for diagnosis and staging of the disease. NEF is dispensable and probably suffers counter-selection during ex vivo viral propagation in vivo. Recent evidence suggests that SIV nef is required for viral propagation in vivo.
vpr
vpr – virion-associated protein of unknown function found in HIV-1, HIV-2, SIVmac, and SIVmnd; typically 15 kD. May be homologous to vpx. Also called “rap” for rapid.
vpu
vpu – protein that promotes extracellular release of viral particles. Found only in HIV-1. Integral membrane phosphoprotein of 16kd; similar to M2 protein of influenza virus. It may be involved in env maturation. It is not found in the virion.
vpx
vpx – virion protein of 12 kD found only in HIV-2 infection. (vpx may have some homology with vpr).
Related research paper:
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
用于酵母繁殖和筛选的培养基
SD是含有盐类、微量元素、维生素、氮源(不含氨基酸的酵母氮碱)和葡萄糖的合成基本培养基。
注意：氮源(不含氨基酸的酵母氮碱)是这种培养基用于筛选的关键，LB，YPD这种复合培养基因为用到了酵母提取物，蛋白胨，基本是氮源全营养的，肯定是不能用来做氮源筛选的
琼脂，学名琼胶，英文名（agar），又名洋菜(agar-agar）、海东菜、冻粉、琼胶、石花胶、燕菜精、洋粉、寒天、大菜丝，是植物胶的一种，常用海产的麒麟菜、石花菜、江蓠等制成，为无色、无固定形状的固体，溶于热水。在食品工业中应用广泛，亦常用作细菌培养基。为什么叫琼脂，主要是用海南的麒麟菜或石花菜制作出来的。海南的简称就是琼。琼脂特性：琼脂的最有用特性是它的凝点和熔点之间的温度相差很大。它在水中需加热至95℃时才开始熔化，熔化后的溶液温度需降到40℃时才开始凝固，所以它是配制固体培养基的最好凝固剂。用琼脂配制的固体培养基，可用以进行高温培养而不熔化，在凝固之前接种时，也不致将培养物烫死。因此，琼脂是制备各种生物培养基中应用最广泛的一种凝固剂。琼脂的浓度，通常是液体培养基的1～1.5%。向左转|向右转
（一）按成分不同划分
1、天然 培养基 (complex medium) 这类培养基含有化学成分还不清楚或化学成分不恒定的天然有机物，也称非化学限定培养基(chemically undefined medium)。牛肉膏蛋白胨培养基和麦芽汁培养基就属于此类。基因克隆技术中常用的LB(Luria—Bertani)培养基也是一种天然培养基，其组成见表5.9。
牛肉浸膏、蛋白胨及酵母浸膏的来源及主要成分
营养物质 牛肉浸膏
来 源 瘦牛肉组织浸出汁浓缩而成的膏状物质
主要成分 富含水溶性糖类、有机氮化合物、维生素、盐等
营养物质 蛋白胨
来 源 将肉、酪素或明胶用酸或蛋白酶水解后干燥而成
主要成分 富含有机氮化合物、也含有一些维生素和糖类的粉末状物质
营养物质 酵母浸膏
来 源 酵母细胞的水溶性提取物浓缩而成的膏状物质
主要成分 富含B类维生素，也含有有机氮化合物和糖类
常用的天然有机营养物质包括牛肉浸膏、蛋白胨、酵母浸膏(表5.10)、豆芽汁、玉米粉、土壤浸液、麸皮、牛奶、血清、稻草浸汁、羽毛浸汁、胡萝卜汁、椰子汁等，嗜粪微生物(coprophilous microorganisms)可以利用粪水作为营养物质。天然培养基成本较低，除在实验室经常使用外，也适于用来进行工业上大规模的微生物发酵生产。
2、合成培养基(synthic medium)是由化学成分完全了解的物质配制而成的培养基，也称化学限定培养基(chemically defined medium)，高氏I号培养基和查氏培养基就属于此种类型。配制合成培养基时重复性强，但与天然培养基相比其成本较高，微生物在其中生长速度较慢，一般适于在实验室用来进行有关微 生物营养需求、代谢、分类鉴定、生物量测定、菌种选育及遗传分析等方面的研究工作。
（二）根据物理状态划分
根据培养基中凝固剂的有无及含量的多少，可将培养基划分为固体培养基、半固体培养基和液体培养基三种类型。
1、固体培养基(so1id medium)
在液体培养基中加入一定量凝固剂，使其成为固体状态即为固体培养基。理想的凝固剂应具备以下条件：①不被所培养的微生物分解利用；②在微生物生长的温度范围内保持固体状态，在培养嗜热细菌时，由于高温容易引起培养基液化，通常在培养基中适当增加凝固剂来解决这一问题；③凝固剂凝固点温度不能太低，否则将不利于微生物的生长；④凝固剂对所培养的微生物无毒害作用；⑤凝固剂在灭菌过程中不会被破坏；⑥透明度好，粘着力强；⑦配制方便且价格低廉。常用的凝固剂有琼脂(agar)、明胶(gelatain)和硅胶(silica gel)。表5.11列出琼脂和明胶的一些主要特征。
对绝大多数微生物而言，琼脂是最理想的凝固剂，琼脂是由藻类(海产石花菜)中提取的一种高度分支的复杂多糖；明胶是由胶原蛋白制备得到的产物，是最早用来作为凝固剂的物质，但由于其凝固点太低，而且某些细菌和许多真菌产生的非特异性胞外蛋白酶以及梭菌产生的特异性胶原酶都能液化明胶，目前已较少作为凝固剂；硅胶是由无机的硅酸钠(Na2SO3)及硅酸钾(K2SiO3)被盐酸及硫酸中和时凝聚而成的胶体，它不含有机物，适合配制分离与培养自养型微生物的培养基。
大家好，我是一个质粒小菜鸟，最近遇到个奇怪的事情，希望大家帮忙看看，究竟出了什么问题。
我构建了一个氨苄抗性的质粒，这个质粒在固体培养基里长出了单菌落，但是我挑菌在液体培养基里无法扩增出来。固体培养基和液体培养基一起配置的，氨苄浓度一致，唯一不同的是固体培养基里加入了琼脂。为了防止是氨苄的问题，我把这个固体培养基上的菌落挑划了新的板，依然能长出菌落。而同一次配置的液体培养基，也能扩增其他质粒，所以我实在想不出是什么其他问题，希望大家帮帮忙。
