Product Specifications:
Item#1010: Recombinant Protease HIV-1 HXB2
Concentration: See vial
Mass/vial: 100ug
Volume/vial: See vial
Diluent: PBS+ <0.01% SDS
Purity: >95%
Stabilizer: None
Preservative: None
Storage: -75°C
Physical State: Frozen Liquid
Stability: Minimum 6 months at -75°C
Application: ELISA, Western ELISA, Immunological Studies, Drug Screening.
Description: Full length HIV-1 endonuclese (protease) expressed in the the E.coli expression system.
Purification: This protein is purified by solvent extraction and preparative electrophoresis to >98% purity, as determined by SDS-PAGE, reduced.
Approx. Molecular Weight:36kD.
Specificity: This protein binds to human anti-protease polyclonal antibodies and murine monoclonal antibodies as determined ELISA and Western ELISA
Biological Activity: Not determined.
Application and Instructions for use
This protein is stabilized against autoproteolysis by the buffer conditions with a pH between 2 - 2.5. To reactivate the protein, dilute with buffers between pH 5.5 - 7.0.
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
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动物组织块冻存在液氮,取出后放到1.5mlEP管中,EP管中加入RIPA裂解液(按照0.1mg组织最多1ml裂解液的体积使用,用前已加入PMSF),使用电动组织研磨器研磨,每次研磨时间10秒左右,研磨过程中发现蛋白质起泡,就像做蛋糕时鸡蛋被打出泡一样。请问如何避免这种情况?
PS,本来试着在冰上操作,但是电动组织研磨器工作时EP管震动的很厉害,没法放在冰上。
是不是可以先放-70冰箱预冷,然后就可以加液氮研磨组织了?
会不会爆裂啊?
1机械法:主要通过机械切力的作用使组织细胞破碎的方法,常用的器械有组织捣碎机、匀浆器、研钵和研磨、压榨器等。
1.1组织捣碎机
将材料配成稀糊状液,放置于筒内约1/3体积,盖紧筒盖,将调速器先拨至最慢处,开动开关后,逐步加速至所需速度。一般用于动物组织、植物肉质种子、柔嫩的叶芽等,转速可高达10000rpm/M以上。由于旋转刀片的机械切力很大,制备一些较大分子如核酸则很少使用。
1.2匀浆器
先将剪碎的组织置于管中,再套入研杆来回研磨,上下移动,即可将细胞研碎。匀浆器的研钵磨球和玻璃管内壁之间间隙保持在十分之几毫米距离。制作匀浆器的材料,除玻璃外,还可以用硬质塑料、不锈钢、人造荧光树脂等。此法细胞破碎程度比高速组织捣碎机为高,适用于量少和动物脏器组织。
存在的问题;较易造成堵塞的团状或丝状真菌,较小的革兰氏阳性首以及有些亚细胞器,质地坚硬,易损伤匀浆阀,也不适合用该法处理。
1.3. 研钵
多用于细菌或其他坚硬植物材料,研磨时常加入少量石英砂,玻璃粉或其他研磨剂,以提高研磨效果。
1.44. 细菌磨
是一种改良了的研磨器,比研钵具有更大的研磨面积,而且低部有出口。操作时先把细菌和研磨粉调成糊状,每次加入一小勺,研磨20-30秒即可将细菌细胞完全磨碎。
2 物理法
主要通过各种物理因素使组织细胞破碎的方法。在生化制备中常用的方法有:
2.1. 反复冻溶法
原理:因突然冷冻,细胞内冰晶的形成及胞内外溶剂浓度的突然改变而破坏细胞。
方法:将待破碎的细胞在-20度以下冰冻,室温融解,反复几次,由于细胞内冰粒形成和剩余细胞液的盐浓度增高引起溶胀,使细胞结构破碎。
特点:此法适用于组织细胞,多用于动物性材料,对微生物细胞作用较差。
2.2. 急热骤冷法
将材料投入沸水中,维持85-90分钟,至水浴中急速冷却,此法可用于细菌及病毒材料。
2.3. 超声波处理
用一定功率的超声波处理细胞悬液,使细胞急剧震荡破裂,此法多适用于微生物材料,用大肠杆菌制备各种酶,常选用50-100毫克菌体/毫升浓度,频高于15~20KHz的超声波在高强度声能输入下可以进行细胞破碎。其破碎机理:可能与空化现象引起的冲击波和剪切力有关。超声破碎的效率与声频、声能、处理时间、细胞浓度及首种类型等因素有关。
行细胞破碎。其破碎机理:可能与空化现象引起的冲击波和剪切力有关。超声破碎的效率与声频、声能、处理时间、细胞浓度及首种类型等因素有关。
特点:操作简单,重复性较好,节省时间;多用于微生物和组织细胞的破碎。
存在问题:超声波破碎在实验室规模应用较普遍,处理少量样品时操作简便,液量损失少,但是超声波产生的化学自由基团能使某些敏感性活性物质变性失活。而且大容量装置声能传递,散热均有困难,应采取相应降温措施。对超声波敏感和核酸应慎用。空化作用是细胞破坏的直接原因,同时会产生活性氧,所以要加一些巯基保护剂。展开
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