Role of oxidative stress in vinorelbine-induced vascular endothelial cell injury

Vinorelbine (VNR), a vinca alkaloid anticancer drug, often causes vascular injury such as venous irritation, vascular pain, phlebitis, and necrotizing vasculitis. The purpose of this study was to identify the mechanisms that mediate the cell injury induced by VNR in porcine aorta endothelial cells (PAECs). PAECs were exposed to VNR for 10 min followed by further incubation in serum-free medium without VNR. The exposure to VNR (0.3–30 μM) decreased the cell viability concentration and time dependently. The incidence of apoptotic cells significantly increased at 12 h after transient exposure to VNR. At the same time, VNR increased the activity of caspases. Interestingly, VNR rapidly depleted intracellular glutathione (GSH) and increased intracellular reactive oxygen species (ROS) production. Moreover, VNR depolarized the mitochondrial membrane potential and decreased cellular ATP levels. These VNR-induced cell abnormalities were almost completely inhibited by GSH and N-acetylcysteine. On the other hand, l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH synthesis, aggravated the VNR-induced loss of cell viability. These results clearly demonstrate that VNR induces oxidative stress by depleting intracellular GSH and increasing ROS production in PAECs, and oxidative stress plays an important role in the VNR-induced cell injury.     

SDS-聚丙烯酰氨凝胶电泳与液质联用技术分离和鉴定小鼠巨噬细胞膜蛋白

用膜蛋白分离试剂盒提取巨噬细胞膜蛋白,然后用SDS-聚丙烯酰氨凝胶电泳进行分离。将每个泳道平均切成8份,合并两个泳道同样位置的胶条,分别进行胶内酶解。酶解得到的多肽经脱盐后进入毛细管反相柱进行反相分离,分离后的肽段直接进入电喷离子源质谱仪进行一级和二级质谱分析。质谱数据用SEQUEST软件对小鼠IPI蛋白数据库进行检索,得到一个含有1000多种蛋白的名单,其中包括458种经GOA注释的膜蛋白。对膜蛋白部分进一步分析发现,其中包括CD11b、TNF-a、F4/80、CD14、CD18、CD86、CD44、CD16、Toll样受体等已知表达在巨噬细胞表面的蛋白分子,还包括另外13种CD分子和18种Ras相关GTPase,除了这些已知蛋白之外,还鉴定出若干新蛋白分子,为进一步深入研究巨噬细胞生物学功能提供了目标分子。     

Phosphate-binding tag, a new tool to visualize phosphorylated proteins

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE).     

The search for the chaperonin 60 receptors

Chaperonin (Cpn)60 proteins have the ability to activate human and murine myeloid cells. There is contradictory evidence that the receptor for this protein is either similar to that of lipopolysaccharide--CD14 and one or other toll-like receptor (e.g. TLR4) or is some other, undidentified, receptor. In an attempt to directly identify the receptor for Mycobacterium tuberculosis Cpn60.1 we have used two approaches. The first is to use Cpn60.1 as an affinity ligand to pull out the receptor from lysates of the murine monocyte cell line RAW 264.7. The second is to crosslink Cpn60.1 to its receptor on RAW cells and isolate the complex by immunoprecipitation. These methods have worked for other receptors. Using affinity chromatography, 2D SDS-PAGE and peptide mass fingerprinting with MALDI-TOF MS it was found that a number of proteins had the ability to bind to Cpn60.1 on an affinity matrix. We identified five proteins, three of which were likely to be on the cell surface. One of these proteins, the endoplasmic reticulum molecular chaperone, BiP did bind to Cpn60.1 with low affinity. Protein crosslinking studies proved inadequate as insufficient protein could be isolated for mass spectrometric identification. Thus, it appears that Cpn60.1, like Hsp70, may bind to a number of cell surface proteins. BiP appears to be one of these receptor proteins but more work is needed to identify those responsible for signalling. Of interest, CD14 and TLR4 were not identified in this study as a receptor for Cpn60.1.     

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).     

Regulation by phorbol esters of asialoglycoprotein and transferrin receptor distribution and ligand affinity in a hepatoma cell line

We have investigated the simultaneous regulation of cell surface distribution and ligand binding of the asialoglycoprotein (ASGP) receptor and the transferrin receptor in a hepatoma cell line by phorbol esters. One hour exposure to phorbol esters causes a redistribution of both receptors to the cell interior as shown by radioligand binding at 4 degrees C and selective immunoprecipitation from the plasma membrane. This effect is temperature- and dose-dependent and is not seen with 4-alpha-phorbol, an inactive tumor promoter. The mechanism and kinetics of the ASGP receptor response to phorbol esters appears to differ from that of the transferrin receptor in this cell line. Within the first 10 min there is a decrease in binding of iodinated ligands for both receptors to the HepG2 cell surface. For the transferrin receptor this results from a net internalization of receptor molecules from the plasma membrane pool, while for the ASGP receptor this decrease is accounted for by a 3.5-fold reduction in ligand binding affinity (6.6 X 10(-8) M to 24.0 X 10(-8) M), with essentially no change in the number of ASGP receptors recoverable from the plasma membrane pool by immunoprecipitation. The altered affinity of the ASGP-R is transient; the Kd returns to control levels by 20 min of continued exposure to the agent. The transferrin receptor shows no change in binding affinity during the course of exposure to phorbol esters. ASGP receptors in cells exposed to phorbol esters for 1 h maintain their competence to deliver exogenous ligand to intracellular sites of degradation and to participate in the recycling pathway of receptor-mediated endocytosis, although at a lower rate than in control cells. We conclude that under identical conditions phorbol esters modulate the binding capacity of two receptors at the cell surface by separate mechanisms. Furthermore, the transient nature of the altered ASGP-R binding affinity suggests that at least two mechanisms, receptor redistribution as well as decreased binding affinity, are operative in the modulation of ASGP-R cell surface binding during the first hour of exposure to the phorbol esters.     

Selective isolation of individual cell surface proteins from tissue culture cells by a cleavable biotin label

A method was developed to isolate cell surface proteins by a simple two-step procedure. Hepatocyte cell surface proteins were labeled by a cleavable biotin derivative in a covalent pulse reaction. Under the described conditions, NHS-SS-biotin proved to be an impermeant, cell surface-specific label which does not affect the impermeant, cell surface-specific label which does not affect the viability of rat hepatocytes. Biotinylated cell surface proteins could be selectively separated under non-denaturing conditions from non-biotinylated proteins and biotin-containing carboxylases by avidin affinity chromatography and sulfhydryl-mediated elution. Subsequent to alkylation of the eluted protein, individual cell surface proteins could be isolated by immunoprecipitation as shown for a selected Mr 120,000 glycoprotein gp120 of the hepatocyte plasma membrane. Using this technique, a transit time of gp120 from the endoplasmic reticulum to the cell surface of 2 h was determined. The results show that the combination of labeling with a cleavable biotin derivative, non-denaturing avidin affinity chromatography and immunoprecipitation is a useful method to isolate and study individual cell surface proteins.     

Mapping the proteome of thylakoid membranes by de novo sequencing of intermembrane peptide domains

The proteome of a membrane compartment has been investigated by de novo sequence analysis after tryptic in gel digestion. Protein complexes and corresponding protein subunits were separated by a 2-D Blue Native (BN)/SDS-PAGE system. The transmembrane proteins of thylakoid membranes from a higher plant (Hordeum vulgare L.) were identified by the primary sequence of hydrophilic intermembrane peptide domains using nano ESI-MS/MS-analysis. Peptide analysis revealed that lysine residues of membrane proteins are primarily situated in the intermembrane domains. We concluded that esterification of lysine residues with fluorescent dyes may open the opportunity to label membrane proteins still localized in native protein complexes within the membrane phase. We demonstrate that covalent labelling of membrane proteins with the fluorescent dye Cy3 allows high sensitive visualization of protein complexes after 2-D BN/SDS-PAGE. We show that pre-electrophoretic labelling of protein subunits supplements detection of proteins by post-electrophoretic staining with silver and CBB and assists in completing the identification of the membrane proteome.     

Labeling of integrin alpha v beta 3 with 58Co(III). Evidence of metal ion coordination sphere involvement in ligand binding.

Integrin-mediated cell adhesion to the extracellular matrix is divalent metal ion-dependent; however, a demonstration of the interaction between native integrins and divalent metal ions is lacking. Here we provide direct evidence that the vitronectin receptor (VNR) is a metalloprotein. The unique electron shell of Co(II), an ion which we show supports ligand recognition by VNR, enables its oxidative conversion to inert Co(III). This property facilitated "affinity labeling" of VNR with 58Co(III) by oxidation of the metal ion in situ (i.e. in position). An average of 3.5 +/- 0.5 mol of cobalt were incorporated per mol of VNR. The ability of VNR to bind metal ions was independently confirmed by examining the interaction between VNR and Mn2+ under native conditions. The apparent high affinity between VNR and Mn2+ allowed us to observe the specific binding between 54Mn2+ and VNR by equilibrium gel filtration studies. Interestingly, the oxidative incorporation of Co(III) into VNR specifically blocked ligand binding, suggesting that the coordination sphere of metal ion bound to VNR is a critical determinant in integrin-ligand recognition. Furthermore, Mn2+ abolished the oxidative affinity labeling of VNR with Co(III) and consequently blocked the inactivation of VNR by in situ incorporation of Co(III). Thus, Mn2+ and Co2+ bind to the same or mutually exclusive sites on VNR. These observations provide the first demonstration that an integrin, specifically VNR, is a metalloprotein and demonstrate a functional link between the coordination sphere of the bound metal ion and ligand recognition by this receptor.     

A proteomic study reveals the diversified distribution of plasma membrane-associated proteins in rat hepatocytes

To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM-associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one-dimensional (1-D) SDS-PAGE or two-dimensional (2-D) benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE before nano-Liquid Chromatography-Electrospray Ionization--tandem mass spectrometry analysis (LC-ESI-MS/MS). A total of 613 non-redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane-associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction-specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1-D SDS-PAGE and 2-D 16-BAC/SDS-PAGE on the separation of integral membrane proteins.     

An analysis of autologous glucocorticoid receptor protein regulation in AtT-20 cells also reveals differential specificity of the BuGR2 monoclonal antibody

When the anti-glucocorticoid receptor monoclonal antibody (BuGR2) was initially incorporated either into a new immunoassay strategy or into a traditional sedimentation analysis technique, both methods failed to reveal any change in the cellular content or distribution of BuGR2-reactive antigen following glucocorticoid treatment of AtT-20 cells. Furthermore, the immunoassay also generated strong positive signals with cytosol and nuclear extracts from a receptor-negative cell line (E8.2) derived from L929 cells. However, when the BuGR2 antibody was incorporated into a combined immunoprecipitation/Western blot analysis of AtT-20 cell extracts, only the glucocorticoid receptor protein produced a signal on the Western blot, even though other proteins had been specifically immunoprecipitated by BuGR2 antibody and were clearly present on the Western blot membrane. Applying the latter approach to AtT-20 cells chronically treated with glucocorticoid, we observed not only that the receptor protein rapidly and persistently (1–96 h) accumulated in the nucleus, but also that its total cellular content was first depleted (24 h) and then was progressively replenished (48–96 h). From these studies in AtT-20 cells we conclude: (i), the BuGR2 antibody can exhibit differential immunospecificity dependent upon whether antigen mixtures are denatured or not; (ii), glucocorticoid receptor protein resided almost exclusively in the nucleus during four days of glucocorticoid treatment and (iii), the same treatment regimen resulted in total receptor protein levels being regulated in a biphasic pattern. Together, these results suggest that receptor regulation in AtT-20 cells is a complex event, and that, since steroid was constantly present during our experiments, other factors are involved in regulation of receptor levels.     

Identification of a lipid-anchored heparan sulfate proteoglycan in Schwann cells

Schwann cells synthesize both hydrophobic and peripheral cell surface heparan sulfate proteoglycans (HSPGs). Previous analysis of the kinetics of radiolabeling suggested the peripheral HSPGs are derived from the membrane-anchored forms (Carey, D., and D. Evans. 1989. J. Cell Biol. 108:1891-1897). Peripheral cell surface HSPGs were purified from phytic acid extracts of cultured neonatal rat sciatic nerve Schwann cells by anion exchange, gel filtration, and laminin-affinity chromatography. Approximately 250 micrograms of HSPG protein was obtained from 2 X 10(9) cells with an estimated recovery of 23% and an overall purification of approximately 2000-fold. SDS-PAGE analysis indicated the absence of non-HSPG proteins in the purified material. Analysis of heparinase digestion products revealed the presence of at least six core protein species ranging in molecular weight from 57,000 to 185,000. The purified HSPGs were used to produce polyclonal antisera in rabbits. The antisera immunoprecipitated a subpopulation of 35SO4- labeled HSPGs that were released from Schwann cells by incubation in medium containing phosphatidylinositol-specific phospholipase C (PI- PLC); smaller amounts of immunoprecipated HSPGs were also present in phytic acid extracts. In the presence of excess unlabeled PI-PLC- released proteins, immunoprecipitation of phytic acid-solubilized HSPGs was inhibited. SDS-PAGE analysis of proteins immunoprecipitated from extracts of [35S]methionine labeled Schwann cells demonstrated that the antisera precipitated an HSPG species that was present in the pool of proteins released by PI-PLC, with smaller amounts present in phytic acid extracts. Nitrous acid degradation of the immunoprecipitated proteins produced a single 67,000-Mr core protein. When used for indirect immunofluorescence labeling, the antisera stained the external surface of cultured Schwann cells. Preincubation of the cultures in medium containing PI-PLC but not phytic acid significantly reduced the cell surface staining. The antisera stained the outer ring of Schwann cell membrane in sections of adult rat sciatic nerve but did not stain myelin or axonal membranes. This localization suggests the HSPG may play a role in binding the Schwann cell plasma membrane to the adjacent basement membrane surrounding the individual axon-Schwann cell units.     

Evidence for Shared Receptor Proteins for Human Interleukin-3 and Granulocyte-Macrophage Colony-Stimulating Factor in the Human M-07 Cell Line

Abstract

The biologic response of the human leukemia cell line M-07 to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 4 (IL-4) is mediated by a low number of high affinity receptors. Cross-competition studies revealed that IL-3 and GM-CSF partially inhibited the specific binding of the heterologous radiolabeled ligand, whereas IL-4 binding was not affected by these cytokines. The molecular mechanism of cross-competition was investigated by chemical crosslinking and immuno-precipitation. Trimolecular receptor complexes consisting of a major 73kDa and two minor 120 and 128kDa membrane proteins for IL-3, and a major 84kDa and two minor 120 and 130 kDa proteins for GM-CSF were found on M-07 cells. The 73 and 84kDa proteins represent distinct and non-linked membrane proteins and are identical with the cloned, low affinity IL-3 and GM-CSF receptor proteins (Gearing et al, 1989, Hayashida et al, 1990). The higher molecular weight proteins share common binding sites as evidenced by immunoprecipitation of double-crosslinked membranes. The 120/128kDa proteins are most likely identical with the recently cloned and shared β-subunit of the IL-3 and GM-CSF receptor (Kitamura et al, 1991) containing a single or two IL-3 and/or GM-CSF molecules.     

Combining enhanced metabolic labeling with immunoblotting to detect interactions of endogenous cellular proteins

Metabolic labeling, immunoblotting and two-dimensional isoelectric focusing/SDS-PAGE are powerful techniques for characterizing endogenously expressed cellular proteins and their interactions. We achieved improved resolution and sensitivity for the detection of metabolically labeled proteins separated on two-dimensional gels by electroblotting the proteins onto polyvinylidene difluoride or nitrocellulose membranes and detecting the 35S signal on a bio-image analyzer. We obtained independent detection of specific proteins from the same blot by subsequent rehydration of the membrane and immunoblot analysis. The combination of these enhanced detection techniques with immunoprecipitation and two-dimensional electrophoresis on precast minigels provides a simple, sensitive method for detecting interactions between endogenous proteins in the cell.     

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (> 95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.     

Design,generation, and testing of mammalian expression modules that tag membrane proteins

The expression of mammalian membrane proteins in laboratory cell lines allows their biological functions to be characterized and carefully dissected. However, it is often difficult to design and generate effective antibodies for membrane proteins in the desired studies. As a result, expressed membrane proteins cannot be detected or characterized via common biochemical approaches such as western blotting, immunoprecipitation, or immunohistochemical analysis, and their cellular behaviors cannot be sufficiently investigated. To circumvent such roadblocks, we designed and generated two sets of expression modules that consist of sequences encoding for three essential components: (1) a signal peptide from human receptor for advanced glycation end products that targets the intended protein to the endoplasmic reticulum for cell surface expression; (2) an antigenic epitope tag that elicits specific antibody recognition; and (3) a series of restriction sites that facilitate subcloning of the target membrane protein. The modules were designed with the flexibility to change the epitope tag to suit the specific tagging needs. The modules were subcloned into expression vectors, and were successfully tested with both Type I and Type III human membrane proteins: the receptor for advanced glycation end products, the Toll‐like receptor 4, and the angiotensin II receptor 1. These expressed membrane proteins are readily detected by western blotting, and are immunoprecipitated by antibodies to their relative epitope tags. Immunohistochemical and biochemical analyses also show that the expressed proteins are located at cell surface, and maintain their modifications and biological functions. Thus, the designed modules serve as an effective tool that facilitates biochemical studies of membrane proteins.     

Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation. Accepted: 10 June 1999     

Easy detection of chromatin binding proteins by the histone association assay

The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and nonchromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes.     

牛肾上腺皮质LDL受体的纯化和抗体的制备

牛肾上腺皮质LDL受体经Triton X-100增溶,DEAE_(32)离子交换柱和LpB Sepharose亲和柱层析,在SDS-PAGE中有三条区带,分别在原点;Mr 160kD;Mr125kD处。进一步用8％SDS-PAGE纯化三个区带的蛋白质分别免疫新西兰大白兔所得的抗体,应用免疫印迹和ECL非同位素标记法可对牛肾上腺皮质和人皮肤纤维细胞膜上的LDL受体进行测定。