A procedure for the purification of beta-lactoglobulin from bovine milk using gel filtration chromatography at low pH

A simple and rapid procedure for the purification of beta-lactoglobulin (β-LG) from bovine milk is described. The procedure exploits the major difference in molecular mass of β-LG and other whey components and the existence of the former in monomeric form at acidic pH. Gel filtration of whey was carried out using a Bio-Gel P10 column at pH 3.0. Residual caseins and other milk proteins were excluded from the gel and β-LG and alpha-lactalbumin (α-LA) emerged as two fully resolved peaks. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that β-LG was purified to apparent homogeneity, while absorption, fluorescence, and circular dichroism spectroscopy indicated the native-like conformation of the protein. Western blot analysis revealed that the antibodies raised against the purified β-LG in rabbits also readily react with the commercial bovine protein. This procedure requires only 4-5 hr for the purification of about 10 mg of β-LG from a single run while using a small column (2.3 cm x 83 cm) of Bio-Gel P10 and has the potential for scaling up.     

草鱼Ⅲ型呼肠孤病毒NS66抗体制备及应用

[背景]草鱼Ⅲ型呼肠孤病毒(grass carp reovirus,GCRV genotypeⅢ) 104株可导致典型性草鱼出血病,对其编码片段的分析有望为临床免疫学检测提供依据。[目的]研究GCRV104株s6基因节段编码蛋白NS66的可能功能,制备良好的GCRV104株NS66蛋白多克隆抗体并分析其特异性。[方法]PCR方法扩增GCRV104株s6基因片段,并克隆至表达载体pGEX-4T-3,转化到大肠杆菌BL21后用IPTG诱导表达,其产物经SDS-PAGE鉴定分析后,通过纯化获得目的蛋白。然后用纯化的pGEX-4T-3-NS66重组蛋白免疫小鼠,获得Anti pGEX-4T-3-NS66多克隆抗体,Western blot测定抗体效价,Western blotting和间接免疫荧光试验(indirect immunofluorescence assay,IFA)鉴定抗体特异性。[结果]SDS-PAGE分析显示表达的重组蛋白约66 kD,大小与预期相符,主要存在于包涵体中;Western blotting测得制备的多克隆抗体效价大于1:50 000,Western blotting和IFA结果表明,制备的多克隆抗体能特异性识别GCRV104病毒。[结论]GCRV104病毒编码的非结构蛋白NS66可能参与了复制和组装过程,形成病毒包涵体,这为建立GCRV104免疫诊断方法及研究GCRV编码的NS66蛋白的功能奠定了前期基础。     

Antibodies against synthetic peptides used to determine the topology and site of glycosylation of the cGMP-gated channel from bovine rod photoreceptors.

Peptides corresponding to amino acids 321-339 (peptide GS21) and 416-431 (peptide GS31) of the cGMP-gated channel from bovine rod photoreceptors were synthesized and used as antigens for the preparation of polyclonal antibodies. After affinity purification, both antipeptide antibodies were found to bind specifically to the channel protein after Western blotting, but only the antibody against GS21 gave satisfactory results on enzyme-linked immunosorbent assay and electron microscopy. Using immunocytochemistry, we were able to localize amino acids 321-339 to the extracellular side of the rod photoreceptor plasma membrane. By synthesizing heptapeptides corresponding to amino acids 324-330 (peptide GS2s) and 420-426 (peptide GS3s), we were able to affinity purify antibodies specific for two N-glycosylation consensus sites in the channel protein. As assessed by Western blotting, antibodies against GS3s were found to bind to both the glycosylated and deglycosylated channel proteins, whereas antibodies against GS2s only bound to the channel protein after enzymatic deglycosylation. Together, these results allow the refinement of folding models for the cGMP-gated channel and implicate Asn-327 as being the sole site of N-glycosylation.     

Prokaryotic expression and polyclonal antibody preparation of a novel Rab-like protein mRabL5

Rab GTPases, which belong to the Ras superfamily, represent a group of small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. We first identified mRabL5 (GenBank Accession No. NP_080349), a novel Mus musculus Rab-like protein, present as a Golgi-associated protein. Here we presented the results of the cloning, prokaryotic expression, purification, and polyclonal antibody production of the novel Rab-like protein. In order to obtain a specific antibody against mRabL5, we prepared two GST fusion proteins, full-length mRabL5 GST fusion protein and mRabL5 C terminus GST fusion protein, to immunize rabbits. Western blot analysis showed that both antibodies prepared against full length of mRabL5 and its C terminus, respectively, can recognize mRabL5 protein. Immunofluorescence of mRabL5 in NIH3T3 cells using the two antibodies showed its perinuclear clustering distribution pattern. The polyclonal antibodies preparation against mRabL5 provided a good tool for us to study the functional involvement of mRabL5.     

Identification of lipoprotein lipase immunoreactive protein in pre- and postheparin plasma from normal subjects and patients with type I hyperlipoproteinemia

Postheparin plasma is a convenient source for the measurement of lipoprotein lipase (LPL) in humans. Previous studies have focused on the measurement of LPL catalytic activity, and have been unable to conveniently measure the LPL protein or identify possibly different plasma forms of the enzyme. Pre- and postheparin plasma was treated with a highly specific antibody raised against bovine milk LPL and the immunoprecipitate was analyzed by Western blotting. In normal subjects there were several species of LPL in plasma. A 56 kD protein increased after heparin injection, and likely represented active LPL. The anti-LPL antibody reacted specifically with this 56 kD protein, and also reacted specifically with proteins at 52 kD, 69 kD, as well as a 20 kD breakdown product. In addition, using peptide mapping, the 56 kD protein was structurally similar to the 52 and 69 kD LPL proteins. The antibodies were affinity purified, biotinylated, and used to quantitate LPL immunoreactive mass using an enzyme-linked immunosorbent assay (ELISA). LPL immunoreactive mass was present in all subjects in preheparin plasma. In postheparin plasma, five patients with type I hyperlipoproteinemia displayed decreased LPL immunoreactive mass when compared to normal subjects, although there was a wide range of specific activity of the small amount of enzyme present. When the LPL from the plasma of the patients was immunoprecipitated and Western blotted, there was considerable heterogeneity in the appearance of the LPL forms, and an overall decrease in LPL protein. Thus, several different immunoreactive LPL proteins were present in pre- and postheparin plasma. In preheparin plasma, as well as in patients with type I hyperlipoproteinemia, there was decreased immunoreactive LPL protein, and the LPL protein that was present was of low specific activity.     

H-Type Bovine Spongiform Encephalopathy

We previously reported that some cattle affected by bovine spongiform encephalopathy (BSE) showed distinct molecular features of the protease-resistant pion protein (PrPres ) in Western blot, with a 1-2 kDa higher apparent molecular mass of the unglycosylated PrPres associated with labelling by antibodies against the 86-107 region of the bovine PrP protein (H-type BSE). By Western blot analyses of PrPres, we now showed that the essential features initially described in cattle were observed with a panel of different antibodies and were maintained after transmission of the disease in C57Bl/6 mice. In addition, antibodies against the C-terminal region of PrP revealed a second, more C-terminally cleaved, form of PrPres (PrPres #2), which, in unglycosylated form, migrated as a ≈ 14 kDa fragment. Furthermore, a PrPres fragment of ≈ 7kDa, which was not labelled by C-terminus-specific antibodies and was thus presumed to be a product of cleavage at both N- and C-terminal sides of PrP protein, was also detected. Both PrPres #2 and ≈ 7 kDa PrPres were detected in cattle and in C57Bl/6 infected mice. These complex molecular features are reminiscent of findings reported in human prion diseases. This raises questions regarding the respective origins and pathogenic mechanisms in prion diseases of animals and humans.     

Development and characterization of cholinephosphotransferase antibody

In the present study, we generated antibodies in rabbits against two synthetic peptides, one based on peptide sequence from yeast CPT cDNA (position 86 to 98 of the amino acid sequence) and the other from our guinea pig CPT cDNA (it corresponds to amino acid positions 119 to 130 according to yeast CPT gene). The antibody titers were measured by both dot blot analysis and ELISA using Keyhole limpets hemocyanin coupled CPT peptides. The CPT antibody recognized a single band by Western blot analysis of proteins from guinea pig liver mitochondria and microsomes. The molecular weight of the protein recognized by Western blot analysis is close to the predicted molecular weight (46 kDa) of yeast CPT. Further analysis revealed that the antibody inhibited CPT activity in both subcellular fractions in a dose dependent manner, thus confirming the specificity of the antibody against both subcellular CPT.     

Taenia saginata: Production and characterization of monoclonal antibodies against Taenia saginata metacestode antigens

Cysticercosis is a major cause of economic loss in bovine production due to meat condemnation. Chemotherapy is being used in Brazilian cattle and a diagnostic test to improve the treatment program is desired. We produced monoclonal antibodies against crude (TAEB) and cyst fluid (TAEF) Taenia saginata metacestode antigens using immunized BALB/c mice. After cell fusion, 10 TAEB and nine TAEF hybrids were selected and cloned resulting in 18 IgG₁ and 32 IgM TAEB clones, and 9 IgG₁ and 9 IgM TAEF clones. Ascites was produced and Western blot testing was performed resulting in reactivity to protein fractions of low molecular weight (<18 kDa), 43, 55, 66 and 100 kDa. The indirect immunofluorescence test, with one monoclonal antibody against crude and one against cyst fluid antigens, recognized antigenic fractions of both the scolex and the bladder wall of metacestodes from naturally infected bovine.     

Monoclonal antibodies against various forms of the adenylyl cyclase catalytic subunit and associated proteins

Monoclonal antibodies against partially purified adenylyl cyclase from bovine brain cortex were raised in mice. Three types of antibody were obtained. Type 1 was specific for the calmodulin-sensitive enzyme. Type II also recognized this enzyme, but recognized the calmodulin-insensitive enzymes from a variety of species and tissues as well. Type I antibodies precipitated their antigens in both the native and denatured forms, while type II strongly favored the denatured forms. Type III antibodies precipitated adenylyl cyclase activity, but as shown by Western blot analysis, were directed against 38-kDa and 45-kDa glycoproteins. The 38-kDa protein was identified as synaptophysin.     

Cloning and expression of a prion protein (PrP) gene from Korean bovine (Bos taurus coreanae) and production of rabbit anti-bovine PrP antibody

A PrP gene, from a Korean bovine, exhibiting a nonsense and a missense polymorphism respectively at nucleotides 576 and 652 has been cloned. The latter resulted in Glu to Lys substitution at amino acid residue 218. After expression and purification of the recombinant bovine PrP (recBoPrP) with Glu218Lys substitution, a polyclonal antibody against this protein was raised. ELISA and Western blot analysis suggested that the recBoPrP obtained in this study had a unique conformation not presented in native PrP(C), and the polyclonal antibody recognized PrP in a conformation dependent manner. These reagents will be valuable tools for studying PrP conformation.     

精子特异性蛋白Cabs1多克隆抗体的制备及多用途验证

制备抗小鼠Cabs1 (Calcium-binding and spermatid-specific protein 1)多克隆抗体,以便进一步利用小鼠模型解析Cabs1蛋白在精子变形与精子功能调控中的功能.以密码子的简并性和偏好性为原则,优化小鼠Cabs1基因编码序列,构建重组表达载体ET-30a(+)/Cabs1,...     

A role for phosphorylation of glycogen synthase kinase-3alpha in bovine sperm motility regulation

The long-term goal of our work is to understand biochemical mechanisms underlying sperm motility and fertility. In a recent study we showed that tyrosine phosphorylation of a 55-kDa protein varied in direct proportion to motility. Tyrosine phosphorylation of the protein was low in immotile compared to motile epididymal sperm. Inhibition or stimulation of motility by high calcium levels or cAMP, respectively, results in a corresponding decrease or increase in tyrosine phosphorylation of the 55-kDa protein. Here we report purification and identification of this motility-associated protein. Soluble extracts from bovine caudal epididymal sperm were subjected to DEAE-cellulose, Affi-Gel blue, and cellulose phosphate chromatography. Tyrosine phosphate immunoreactive fractions contained glycogen synthase kinase-3 (GSK-3) activity, suggesting a possible correspondence between these proteins. This suggestion was verified by Western blot analyses following one-dimensional and two-dimensional gel electrophoresis of the purified protein using monoclonal and affinity-purified polyclonal antibodies against the catalytic amino-terminus and carboxy-terminus regions of GSK-3. Further confirmation of the identity of these proteins came from Western blot analysis using antibodies specific to the tyrosine phosphorylated GSK-3. Using this antibody, we also showed that GSK-3 tyrosine phosphorylation was high in motile compared to immotile sperm. Immunocytochemistry revealed that GSK-3 is present in the flagellum and the anterior portion of the sperm head. These data suggest that GSK-3, regulated by phosphorylation, could be a key element underlying motility initiation in the epididymis and regulation of mature sperm function.     

Impact of immunization technology and assay application on antibody performance--a systematic comparative evaluation

Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost ("DNA immunization"; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.     

A new monoclonal antibody, 4-1a,that binds to the amino terminus of human lipoprotein lipase

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5–25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL–GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.     

Production and characterization of a monoclonal antibody to dopamine D2 receptor: comparison with a polyclonal antibody to a different epitope.

A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 microM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein.     

A photoactivatable probe for the Na+/H+ exchanger cross-links a 66-kDa renal brush border membrane protein

Earlier studies on LLC-PK1 cells have demonstrated two pharmacologically distinct Na+/H+ exchangers in renal epithelia. In addition, the cDNA clone for the human Na+/H+ antiporter which is growth factor activatable has been isolated and expressed (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280). We report here the synthesis of an amiloride analogue that can be photoactivated and labeled with 125I. This analogue covalently cross-links a 66-kDa protein of bovine renal brush border membranes. A rabbit polyclonal antibody that was directed against a 20-amino acid peptide of the cytoplasmic domain of its human Na+/H+ antiporter also gives a positive Western against 66-kDa protein of bovine brush border membranes. Thus, the photoactive probe may be helpful in the isolation and purification of the brush border Na+/H+ exchanger.     

A PROCEDURE FOR THE PURIFICATION OF BETA-LACTOGLOBULIN FROM BOVINE MILK USING GEL FILTRATION CHROMATOGRAPHY AT LOW pH

A simple and rapid procedure for the purification of beta-lactoglobulin (β-LG) from bovine milk is described. The procedure exploits the major difference in molecular mass of β-LG and other whey components and the existence of the former in monomeric form at acidic pH. Gel filtration of whey was carried out using a Bio-Gel P10 column at pH 3.0. Residual caseins and other milk proteins were excluded from the gel and β-LG and alpha-lactalbumin (α-LA) emerged as two fully resolved peaks. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that β-LG was purified to apparent homogeneity, while absorption, fluorescence, and circular dichroism spectroscopy indicated the native-like conformation of the protein. Western blot analysis revealed that the antibodies raised against the purified β-LG in rabbits also readily react with the commercial bovine protein. This procedure requires only 4–5 hr for the purification of about 10 mg of β-LG from a single run while using a small column (2.3 cm × 83 cm) of Bio-Gel P10 and has the potential for scaling up.     

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.     

Overexpression and purification of an immunologically reactive His-BIV capsid fusion protein

The gene of the capsid protein of bovine immunodeficiency virus (BIV) was linked to a sequence encoding for six histidines and expressed as the (His)6 p26 capsid fusion protein. The fusion protein was strongly expressed as both soluble and insoluble forms after induction by isopropylthio-beta-d-galactoside. Purification was based on interaction of the hexa-histidine polypeptide with metal ions. Expression could represent 11% of the total protein in Escherichia coli, allowing more than 20 mg of highly purified protein to be obtained per liter of bacterial culture. The (His)6 p26 capsid fusion protein purified by immobilized metal affinity chromatography reacted specifically in Western blot with sera from cattle experimentally infected by BIV, as well as with two monoclonal antibodies directed against different epitopes of the Gag protein. The ease of expression, purification, and specificity of this fusion protein should permit a thorough study of prevalence of BIV infection in large-scale serological studies of field samples.     

Peptides from the SARS-associated coronavirus as tags for protein expression and purification

Protein tagging with a peptide is a commonly used technique to facilitate protein detection and to carry out protein purification. Flexibility with respect to the peptide tag is essential since no single tag suites all purposes. This report describes the usage of two short peptides from the SARS-associated coronavirus nucleocapsid (SARS-N) protein as protein tags. Plasmids for the generation of tagged proteins were generated by ligating synthetic oligonucleotides for the peptide-coding regions downstream of the protein coding sequence. The data show recognition of prokaryotically expressed HIV-1 Gag/p24 fusion protein by Western blot and efficient affinity purification using monoclonal antibodies against the tags. The SARS peptide antibody system described presents an alternative tagging opportunity in the growing field of protein science.