Product Specifications:
Item# 1005 : Recombinant p17 HIV-1 Core Protein
Concentration: See vial
Mass/vial: See vial
Volume/vial: 1ml
Diluent: MES Buffer, pH. 6.5
Purity: >95%
Stabilizer: None
Preservative: None
Storage: -75°C
Physical State: Frozen Liquid
Stability: At least 24 months at -75°C.
Application: ELISA, Western ELISA, Diagnostics
Description: Recombinant HIV-1 p17 is expressed as a Glutathione S-transferase (GST) fusion protien produced in the E.coli expression system.
Purification: This protein is purified by GST-agarose affinity chromotography and p17 is cleaved from the fusion protein by using Factor Xa. GST is removed from the mixture by a second passage through GST-agarose.
Specificity: This protein binds to HIV-1 positive human serum polyclonal antibodies in ELISA and Western ELISA.
Biological Activity: Recombinant p17 core protein contains authentic N and C termini which is normally processed from the sequence of the matrix antigen with relevant HIV-1 protease.
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. Dilute p17 stock solution in MES buffer immediately before use. ELISA assays may be performed with p17 in solid phase in 10-100ng range. Western ELISA may be performed in 100-500ng depending on the specificity of the primary antibody used in the test system.
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:
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二、限制性内切酶消化样品DNA
三、凝胶电泳分离消化产物
四、如果靶序列>5kb，在0.25M的HCl中进行震荡脱嘌呤10min，ddH2O洗一次
五、用变性液震荡处理30min，ddH2O洗一次
六、中和液震荡处理30min
七、裁取合适大小的尼龙膜或硝酸纤维素膜进行转膜操作，可用真空转膜仪或者搭滤纸桥，需要20×SSC
八、制备探针，可用地高辛标记（以地高辛为例）
九、将膜放入杂交瓶，42°C预杂交30min
十、倒掉预杂交液，加入杂交液，适当的温度进行杂交4h至过夜
十一、洗膜，先用2×SSC+0.1%SDS，20-25°C洗2×5min，再用0.5×SSC+0.1%SDS洗2×15min。然后用washing buffer洗1-5min，接着用blocking solution洗30min后，用antibody solution洗30min，再用washing buffer洗2×15min，再用detection buffer洗2-5min后，取出膜，放于保鲜膜上，在结合有DNA的一面滴加CSPD ready-to-use后，立刻盖上保鲜膜，让CSPD ready-to-use均匀的布满膜表面，室温放置5min后，37°C温育10min以上
十二、放射自显影，可用成像系统信号累积模式显影或用X-ray显影
