Blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and de...Blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and detecting the levels of specific proteins through reactions with primary and secondary antibodies. The state-of-the-art of Western blotting usually generates one blotting membrane per gel. However, multiple copies of blots are useful in many applications. Two blotting copies from a single protein gel, for instance, can be used for identifying a total amount of proteins of interest as well as its specific subpopulation level such as a phosphorylated isoform. To achieve this multi-blotting operation from a single gel, we modified a blotting procedure and developed a novel blotting device. The device consisted of a multi-anode plate and a microcontroller. It was designed to generate a well-controlled electrophoretic voltage profile, which allowed a quasi-uniform transfer of proteins of any size. The prototype device was built and its operation procedure was described. The experimental results clearly supported the notion that the described device was able to achieve multiple blotting from a single gel and reduce time and cost for protein analysis.展开更多
Quantitative real-time PCR （qRT-PCR） has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have b...Quantitative real-time PCR （qRT-PCR） has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have been raised over the accuracy of qRT-PCR in China as well as in the rest of the world. We have previously used qRT-PCR to study the response of ANR1 and other root-expressed MADS-box genes to fluctuations in the supply of nitrate, phosphate and sulphate under hydroponic growth conditions. In this study, we have used both Northern blotting and qRT-PCR analyses to confirm the nutritional regulation of MADS-box genes in Arabidopsis thaliana and test whether both technologies produce the same results. The information obtained indicated that the qRT-PCR results are consistent with those obtained by Northern blotting hybridization for all the tested root-expressed MADS-box genes, in response to different nitrate, phosphate and sulphate growth conditions. Furthermore, our novel results showed that the expressions of AGL12, AGL18, and AGL19 were all down regulated in response to S and P re-supply in both qRT-PCR and Northern blotting analyses.展开更多
Recently, serum Golgi protein 73 （GP73） levels have been found to be elevated in patients with hepatocellu- lar carcinoma （HCC）, and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patien...Recently, serum Golgi protein 73 （GP73） levels have been found to be elevated in patients with hepatocellu- lar carcinoma （HCC）, and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent as- say （ELISA）. Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody （mAb） designated as 6A2 using recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls （P = 0.0036）. Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls （P = 0.0172）.展开更多
Blotting was used to observe cell structures of leaf epidermis cells, and the key method of leaf transaction observation was paraffin section. The concentration, suitable solidification time, melting temperature of ge...Blotting was used to observe cell structures of leaf epidermis cells, and the key method of leaf transaction observation was paraffin section. The concentration, suitable solidification time, melting temperature of gelatin solution and the stain for the gelatin blotting were studied in this research. The results showed that the gelatin blotting could be used to study leaf transaction, it was benefit to make operation easily, and save time, money and so on展开更多
LeSPL-CNR is a crucial transcription factor for fruit ripening of Solanum lycopersicum. The cnr (colorless non-ripening) epimutation resulted from hypermethylation in a 286 bp region of LeSPL-CNR promoter inhibits nor...LeSPL-CNR is a crucial transcription factor for fruit ripening of Solanum lycopersicum. The cnr (colorless non-ripening) epimutation resulted from hypermethylation in a 286 bp region of LeSPL-CNR promoter inhibits normal fruit ripening. In present study, potential regulators of LeSPL-CNR, which could bind to the specific 286 bp region, were screened via south-western blotting and yeast one-hybrid (Y1H) library screening system. Results indicated that a total of 13 and 19 candidate proteins were acquired respectively, and both ribulose-1,5-bisphosphate carboxylase/oxygenase and 40S ribosomal protein were identified by two methods. These would provide some information for revealing roles of DNA methylation and the regulatory mechanism for LeSPL-CNR.展开更多
Plastic resins are known to cause occupational allergies. Therefore, serum-specific antibodies against plastic resins have been widely investigated as diagnostic markers for occupational allergies. In this study, we a...Plastic resins are known to cause occupational allergies. Therefore, serum-specific antibodies against plastic resins have been widely investigated as diagnostic markers for occupational allergies. In this study, we aimed to establish a convenient method for detection of multiple chemical-specific IgG antibodies in human serum based on dot blot analysis. Toluene diisocyanate (TDI), phthalic anhydride (PA), and formaldehyde (FA), which are frequently used to synthesize various resins, reacted well with lysine residues of human serum albumin (HSA) under alkaline conditions. Native polyacrylamide gel electrophoresis (PAGE) showed that the structures of chemical adducts of HSA were different from those of native HSA. Therefore, we performed dot blot assays using these adducts as artificial antigens. Serum samples from workers at plants utilizing plastic resins strongly reacted with TDI, PA, and FA adducts in HSA, while reduced signals were detecting using the serum from unexposed workers. These results suggested that dot blot assays using chemical-HSA adducts as antigens could be beneficial for simultaneously measuring multiple chemical-specific IgGs.展开更多
In this work, Staphylococcus epidermidis (S. epidermidis) was used to prepare the fermentation broths with antioxidant activity. Through the optimization of the carbon source, three kinds of S. epidermidis fermentatio...In this work, Staphylococcus epidermidis (S. epidermidis) was used to prepare the fermentation broths with antioxidant activity. Through the optimization of the carbon source, three kinds of S. epidermidis fermentation broth were obtained and designated as SFB, Gly-SFB, and Glu-SFB, which were cultivated in beef protein medium and the beef protein medium supplemented with glycerol or glucose, respectively. The differences in antioxidant efficacy of SFB, Gly-SFB and Glu-SFB were investigated by evaluating intracellular ROS fluorescence intensity, SOD enzyme activity and MDA concentration. Gly-SFB and Glu-SFB exhibited a greater capacity to eliminate ROS as compared to that of SFB. The intracellular SOD enzyme activity increased as the concentrations of SFB and Gly-SFB increased. Nevertheless, the intracellular SOD enzyme activity was the highest after the treatment with Glu-SFB at the low concentrations. The intracellular MDA content reached a lower value after the treatment with Gly-SFB and Glu-SFB at lower concentrations, which was opposite to the case after the treatment with SFB. WB indicated that the S. epidermidis fermentation broth regulated the expression of relevant proteins in the Nrf2-Keap1 signaling pathway to exhibit the antioxidant effects. This indicates that the S. epidermidis fermentation broth promotes the expression of relevant proteins in the Nrf2-Keap1 signaling pathway, consequently, antioxidant benefits were exerted. The fermentation broth that were prepared by incorporating glycerol or glucose into the culture medium can augment their antioxidant activity.展开更多
The inflammatory response is a crucial physiological process that can lead to tissue damage and is considered a causative factor for various chronic diseases,such as rheumatoid arthritis.Recent research has focused on...The inflammatory response is a crucial physiological process that can lead to tissue damage and is considered a causative factor for various chronic diseases,such as rheumatoid arthritis.Recent research has focused on exploring valuable nutrients derived from Cannabis sativa L.(hemp)seeds,particularly hemp seed proteins.Therefore,this study aimed to investigate the release of anti-inflammatory peptides from Lactobacillus paraplantarum-fermented hemp seed proteins.To confirm the complete hydrolysis of hemp seed proteins during the fermentation process,sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)was employed.Further,the isolation and purification of peptides were achieved through ultrafiltration.The identity of peptides was nextly established using ultra-high performance liquid chromatography coupled with hybrid quadrupole time-of-flight mass spectrometry(UHPLC-QTOF-MS).The results revealed a total of 39 identified peptides in fermented hemp seeds,with 9 peptides selected based on their relative quantity.Notably,AAELIGVP(P1),AAVPYPQ(P2),VFPEVAP(P4),DVIGVPLG(P6),and PVPKVL(P9)demonstrated strong anti-inflammatory abilities in lipopolysaccharide(LPS)-induced RAW264.7 macrophage cells.Molecular docking was used to understand the potential anti-inflammatory mechanism of these 5 peptides,and in silico results indicated that P1,P2,P4,P6,and P9 could bind to the active sites of toll-like receptor 4(TLR-4),nuclear factor-κB(NF-κB),and inhibitor of NF-κB kinase(IKK)with higher binding energies.Overall,these findings indicate that hemp seeds have potential to be a source of bioactive peptides for functional foods with anti-inflammatory properties.展开更多
Background Recombinant erythropoietin(rEPO)is commonly used in therapy but may be abused in sports to enhance endurance.In doping analysis,rEPO can be detected in human urine or blood samples at picogram(pg)levels bas...Background Recombinant erythropoietin(rEPO)is commonly used in therapy but may be abused in sports to enhance endurance.In doping analysis,rEPO can be detected in human urine or blood samples at picogram(pg)levels based on its slightly higher molecular weight(MW)than that of endogenous EPO using western blotting(WB).However,a type of variant erythropoietin(VAR-EPO)encoded by the EPO c.577del variant has a similar MW to rEPO,and these 2 molecules cannot be distinguished using conventional analytical methods.A fit-for-purpose method needs to be developed immediately.Methods In this study,we introduced a reverse–normal immunopurification technique for sample pretreatment to remove VAR-EPO from samples to eliminate its interference with rEPO detection.Firstly,a rabbit monoclonal antibody(mAb)that can specifically recognize trace amounts of VAR-EPO with high affinity was generated.Then,using this antibody to enrich VAR-EPO,we developed reverse–normal immunopurification coupled with WB on the purpose of analyzing rEPO in urine and serum samples.Next,the method was fully validated and evaluated using blank samples,spiked samples and rEPO excreted samples.Finally,the identification criteria of rEPO was established.Results A specific anti-VAR mAb with high affinity was developed.Using it,we developed the doping analytical method for rEPO.Our method effectively detects and removes VAR-EPO,enabling accurate rEPO detection.Conclusion A method has already been applied for rEPO confirmation in routine doping analyses.展开更多
文摘Blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and detecting the levels of specific proteins through reactions with primary and secondary antibodies. The state-of-the-art of Western blotting usually generates one blotting membrane per gel. However, multiple copies of blots are useful in many applications. Two blotting copies from a single protein gel, for instance, can be used for identifying a total amount of proteins of interest as well as its specific subpopulation level such as a phosphorylated isoform. To achieve this multi-blotting operation from a single gel, we modified a blotting procedure and developed a novel blotting device. The device consisted of a multi-anode plate and a microcontroller. It was designed to generate a well-controlled electrophoretic voltage profile, which allowed a quasi-uniform transfer of proteins of any size. The prototype device was built and its operation procedure was described. The experimental results clearly supported the notion that the described device was able to achieve multiple blotting from a single gel and reduce time and cost for protein analysis.
基金supported by the Fundamental Research Funds for the Central Universities of China(2009QNA6023)the International Scientific and Technological Cooperation Project of Ministry of Science and Technology of China (2010DFA34430)
文摘Quantitative real-time PCR （qRT-PCR） has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have been raised over the accuracy of qRT-PCR in China as well as in the rest of the world. We have previously used qRT-PCR to study the response of ANR1 and other root-expressed MADS-box genes to fluctuations in the supply of nitrate, phosphate and sulphate under hydroponic growth conditions. In this study, we have used both Northern blotting and qRT-PCR analyses to confirm the nutritional regulation of MADS-box genes in Arabidopsis thaliana and test whether both technologies produce the same results. The information obtained indicated that the qRT-PCR results are consistent with those obtained by Northern blotting hybridization for all the tested root-expressed MADS-box genes, in response to different nitrate, phosphate and sulphate growth conditions. Furthermore, our novel results showed that the expressions of AGL12, AGL18, and AGL19 were all down regulated in response to S and P re-supply in both qRT-PCR and Northern blotting analyses.
基金supported by the Science and Technology Foundation of Nanjing Medical University (No. 07NMUZ005).
文摘Recently, serum Golgi protein 73 （GP73） levels have been found to be elevated in patients with hepatocellu- lar carcinoma （HCC）, and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent as- say （ELISA）. Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody （mAb） designated as 6A2 using recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls （P = 0.0036）. Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls （P = 0.0172）.
基金Supported by Committee of Nature Science Foundation of Heilongjiang Province (C2005-32)Post-doctoral Science Committee of China (LRB04-217)Scientific Research Start Committee of Northeast Agricultural University
文摘Blotting was used to observe cell structures of leaf epidermis cells, and the key method of leaf transaction observation was paraffin section. The concentration, suitable solidification time, melting temperature of gelatin solution and the stain for the gelatin blotting were studied in this research. The results showed that the gelatin blotting could be used to study leaf transaction, it was benefit to make operation easily, and save time, money and so on
文摘LeSPL-CNR is a crucial transcription factor for fruit ripening of Solanum lycopersicum. The cnr (colorless non-ripening) epimutation resulted from hypermethylation in a 286 bp region of LeSPL-CNR promoter inhibits normal fruit ripening. In present study, potential regulators of LeSPL-CNR, which could bind to the specific 286 bp region, were screened via south-western blotting and yeast one-hybrid (Y1H) library screening system. Results indicated that a total of 13 and 19 candidate proteins were acquired respectively, and both ribulose-1,5-bisphosphate carboxylase/oxygenase and 40S ribosomal protein were identified by two methods. These would provide some information for revealing roles of DNA methylation and the regulatory mechanism for LeSPL-CNR.
文摘Plastic resins are known to cause occupational allergies. Therefore, serum-specific antibodies against plastic resins have been widely investigated as diagnostic markers for occupational allergies. In this study, we aimed to establish a convenient method for detection of multiple chemical-specific IgG antibodies in human serum based on dot blot analysis. Toluene diisocyanate (TDI), phthalic anhydride (PA), and formaldehyde (FA), which are frequently used to synthesize various resins, reacted well with lysine residues of human serum albumin (HSA) under alkaline conditions. Native polyacrylamide gel electrophoresis (PAGE) showed that the structures of chemical adducts of HSA were different from those of native HSA. Therefore, we performed dot blot assays using these adducts as artificial antigens. Serum samples from workers at plants utilizing plastic resins strongly reacted with TDI, PA, and FA adducts in HSA, while reduced signals were detecting using the serum from unexposed workers. These results suggested that dot blot assays using chemical-HSA adducts as antigens could be beneficial for simultaneously measuring multiple chemical-specific IgGs.
文摘In this work, Staphylococcus epidermidis (S. epidermidis) was used to prepare the fermentation broths with antioxidant activity. Through the optimization of the carbon source, three kinds of S. epidermidis fermentation broth were obtained and designated as SFB, Gly-SFB, and Glu-SFB, which were cultivated in beef protein medium and the beef protein medium supplemented with glycerol or glucose, respectively. The differences in antioxidant efficacy of SFB, Gly-SFB and Glu-SFB were investigated by evaluating intracellular ROS fluorescence intensity, SOD enzyme activity and MDA concentration. Gly-SFB and Glu-SFB exhibited a greater capacity to eliminate ROS as compared to that of SFB. The intracellular SOD enzyme activity increased as the concentrations of SFB and Gly-SFB increased. Nevertheless, the intracellular SOD enzyme activity was the highest after the treatment with Glu-SFB at the low concentrations. The intracellular MDA content reached a lower value after the treatment with Gly-SFB and Glu-SFB at lower concentrations, which was opposite to the case after the treatment with SFB. WB indicated that the S. epidermidis fermentation broth regulated the expression of relevant proteins in the Nrf2-Keap1 signaling pathway to exhibit the antioxidant effects. This indicates that the S. epidermidis fermentation broth promotes the expression of relevant proteins in the Nrf2-Keap1 signaling pathway, consequently, antioxidant benefits were exerted. The fermentation broth that were prepared by incorporating glycerol or glucose into the culture medium can augment their antioxidant activity.
基金the 4^(th) Brain Korea(BK)21 Plus Project(4299990913942)financed by the Korean Government,Republic of Korea.
文摘The inflammatory response is a crucial physiological process that can lead to tissue damage and is considered a causative factor for various chronic diseases,such as rheumatoid arthritis.Recent research has focused on exploring valuable nutrients derived from Cannabis sativa L.(hemp)seeds,particularly hemp seed proteins.Therefore,this study aimed to investigate the release of anti-inflammatory peptides from Lactobacillus paraplantarum-fermented hemp seed proteins.To confirm the complete hydrolysis of hemp seed proteins during the fermentation process,sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)was employed.Further,the isolation and purification of peptides were achieved through ultrafiltration.The identity of peptides was nextly established using ultra-high performance liquid chromatography coupled with hybrid quadrupole time-of-flight mass spectrometry(UHPLC-QTOF-MS).The results revealed a total of 39 identified peptides in fermented hemp seeds,with 9 peptides selected based on their relative quantity.Notably,AAELIGVP(P1),AAVPYPQ(P2),VFPEVAP(P4),DVIGVPLG(P6),and PVPKVL(P9)demonstrated strong anti-inflammatory abilities in lipopolysaccharide(LPS)-induced RAW264.7 macrophage cells.Molecular docking was used to understand the potential anti-inflammatory mechanism of these 5 peptides,and in silico results indicated that P1,P2,P4,P6,and P9 could bind to the active sites of toll-like receptor 4(TLR-4),nuclear factor-κB(NF-κB),and inhibitor of NF-κB kinase(IKK)with higher binding energies.Overall,these findings indicate that hemp seeds have potential to be a source of bioactive peptides for functional foods with anti-inflammatory properties.
基金WADA and Beijing Sport University for funding under grant numbers 22B06XZ and 2022YB011
文摘Background Recombinant erythropoietin(rEPO)is commonly used in therapy but may be abused in sports to enhance endurance.In doping analysis,rEPO can be detected in human urine or blood samples at picogram(pg)levels based on its slightly higher molecular weight(MW)than that of endogenous EPO using western blotting(WB).However,a type of variant erythropoietin(VAR-EPO)encoded by the EPO c.577del variant has a similar MW to rEPO,and these 2 molecules cannot be distinguished using conventional analytical methods.A fit-for-purpose method needs to be developed immediately.Methods In this study,we introduced a reverse–normal immunopurification technique for sample pretreatment to remove VAR-EPO from samples to eliminate its interference with rEPO detection.Firstly,a rabbit monoclonal antibody(mAb)that can specifically recognize trace amounts of VAR-EPO with high affinity was generated.Then,using this antibody to enrich VAR-EPO,we developed reverse–normal immunopurification coupled with WB on the purpose of analyzing rEPO in urine and serum samples.Next,the method was fully validated and evaluated using blank samples,spiked samples and rEPO excreted samples.Finally,the identification criteria of rEPO was established.Results A specific anti-VAR mAb with high affinity was developed.Using it,we developed the doping analytical method for rEPO.Our method effectively detects and removes VAR-EPO,enabling accurate rEPO detection.Conclusion A method has already been applied for rEPO confirmation in routine doping analyses.
