SDS-PAGE and Dot Blot Autoradiography: Tools for Quantifying Histidine Kinase Autophosphorylation

2019 ◽  
pp. 37-49
Author(s):  
Jonathan T. Fischer ◽  
Ilana Heckler ◽  
Elizabeth M. Boon
Keyword(s):  
Histidine Kinase ◽  
Dot Blot ◽  
Sds Page

scholarly journals THE EFFECT OF FORMALDEHYDE EXPOSURE AND YOGURT SUPPLEMENTATION ON PROFILE AND CHARACTER OF HEPAR TISSUE PROTEIN OF RATS (Rattus norvegicus)

2010 ◽  
Vol 10 (1) ◽  
pp. 132-137
Author(s):  
Chanif Mahdi ◽  
Aulaniam Aulaniam
Keyword(s):  
Rattus Norvegicus ◽  
Specific Protein ◽  
The Body ◽  
Oxygen Species ◽  
Dot Blot ◽  
Laboratory Methods ◽  
Carcinogenic Substance ◽  
Sds Page ◽  
Formaldehyde Exposure ◽  
Specific Band

Formaldehyde is a simplest organic compound of aldehyde or alkanal group. Formaldehyde is a toxic and carcinogenic substance. Formaldehyde contamination through food or feeding diet continuously is very dangerous for the body, especially for bodies organ for instances likes hepar and kidney. Because formaldehyde is sources of reactive oxygen species (ROS) and free radicals substances for the body. This purpose of the study is to know the effect of formaldehyde exposure and yogurt supplementation on profile and characters of rats (Rattus norvegicus) protein hepar tissues. The research methods is laboratory methods. The protein profiles determined by electrophoresis (SDS-PAGE) methods. The character of hepar protein tissue determined by ELISA, Dot Blot and Western Blot methods. The result showed that formaldehyde exposure through the feeding diet of rats affect on profile of hepar protein tissue, that characterized by appear of new band of specific protein with molecule weigh is 29.6 kDa (PSForm 29.6). Yogurt supplementation on rat that exposure by formaldehyde through the feeding diet of rat, that characterized by expressing of new band of specific protein with relative molecule weight 24.8 kDa (PSYogh 24.8 kDa), and followed by depressed or dispear of protein specific band of 29.6 kDa(PSForm 29.6 kDa). The result showed that isolated protein PSForm 29.6 kDa have a antigenecity character.   Keywords: Formaldehyde exposure, yogurt, ROS, profile and protein character

scholarly journals The Reactivity and Allergenic Potential of Hazelnut Peptides

2013 ◽  
Vol 70 (1) ◽  
pp. 25
Author(s):  
Lavinia Florina Calinoiu ◽  
Dan Cristian Vodnar ◽  
Carmen Socaciu
Keyword(s):  
Proteolytic Enzymes ◽  
Peptide Fragments ◽  
Dot Blot ◽  
Specific Antibodies ◽  
Allergenic Potential ◽  
Sds Page ◽  
Allergenic Proteins ◽  
Life Threatening ◽  
Specific Proteins ◽  
Sensitive Method

The aim of this paper was to focus on proteins present in some food products, like hazelnuts and to investigate their allergenic potential. Several techniques were used to characterize these extracted proteins, with respect to their composition, degradability by digestive proteolytic enzymes and their reactivity with specific antibodies. It was important to analyse which proteins were present in the hazelnuts, to see if there were proteins present to trigger an allergic reaction and if the digestion enzymes trypsin and pepsin influence the presence of the (allergic) protein compounds. Allergies to tree nuts and seeds can cause life-threatening and sometimes fatal reactions. To examine the properties of Hazelnut protein it was important to solubilize it by extraction. After extraction, it was investigated how hazelnut protein can be modified by proteases and what the effect was on the immune reaction. The Bradford method is a fast and sensitive method to determine the concentration of soluble protein. When the Bradford reagent (Coomassie Brilliant Blue) binds to the protein, the colour changes from red to purple and the absorption maximum changes from 495 to 595 nm. The value obtained as the final concentration of proteins was 7.3495. SDS-PAGE is a method to separate mixtures of proteins by electrophoresis. Protein molecules are negatively charged by binding of SDS molecules; subsequently they are separated in an electric field. Their differences in size (molecular weight) leads to separation. In this case the method is used to follow proteolytic degradation of hazelnut proteins (allergens) by intestinal proteases (trypsin, pepsin). A different, more specific and sensitive method is immunoblotting (Western Blot) in which the SDS-PAGE separated proteins are transferred from the gel to a membrane and specific antibodies are used in a series of reactions to visualize specific allergens on this membrane. The remarked spots represented a positive identification of allergenic proteins. This means that peptide fragments of various size, produced during the digestion of a protein can still be immunological active. As it was shown there was still reactivity between proteins and specific antibodies. The Dot Blot is a simple immunoblotting technique used to detected specific proteins in a mixture of different proteins and/or other molecules. No separation technique prior to the actual immuno-detection is necessary. Also, Dot Blot confirmed the presence of allergenic proteins made visible through the light spots on the membrane.

scholarly journals The Potential of Candida Biofilm Protein as Bioreceptor for Candidiasis Immunoassay

2018 ◽  
Vol 14 (1) ◽  
pp. 47 ◽  
Author(s):  
Masfufatun Masfufatun ◽  
Loo Haryanto ◽  
Harsono Harsono
Keyword(s):  
Molecular Weight ◽  
Candida Albicans ◽  
Polyclonal Antibody ◽  
Control Group ◽  
Potential Candidate ◽  
Dot Blot ◽  
Protein Extract ◽  
Treatment Groups ◽  
Sds Page ◽  
Mechanical Methods

Abstract: Candidiasis or infection that is caused by Candida has become a new list of the therapeutical problems recently. The difficulties in diagnosing are the main cause of the unsatisfactory results from common therapies and diagnosis methods. This has urged researchers to find alternative ways in candidiasis diagnosis such as serology-based detection using antigen or antibody development. The aim of this study was to evaluate the potential of protein derived from Candida albicans biofilm as bioreceptor on candidiasis immunoassay through Dot Blot method. The research method used descriptive method with the following stages: (1) preparation of Candida albicans biofilm (2) extraction of Candida albicans protein through enzymatic and mechanical methods, (3) determination of protein molecular weight with SDS-PAGE (4) production of polyclonal anti- candida and (5) analysis of protein extract as bioreeceptor on dot blot. Profile of biofilm proteins on SDS-PAGE analysis were shown on molecular weight 27,42; 29,89; 38,10; 44,90; 48,75; 52,92; 55,14; 59,86; 70,56; 87,36; 102,54;115,05; 130,14;143,14;181,53 kD. There were differences in the intensity of dots in the control group (44070) and treatment groups (63170.5). It is noticeable that biofilm protein extract of C. albicans can be used for induction of anti-Candida polyclonal antibody production as the potential candidate of bioreceptor in candidiasis immunoassay. Keywords: SDS-PAGE, polyclonal antibody, immunoassay, dot blot, biofilm

scholarly journals Immunogenity of Protein Extract from Salivary Gland of Anopheles aconitus in Malaria Endemic Area

2017 ◽  
Vol 18 (1) ◽  
pp. 25
Author(s):  
Mahful Septiawan ◽  
Budayatin Budayatin ◽  
Hidayat Teguh Wiyono ◽  
Kartika Senjarini
Keyword(s):  
Salivary Gland ◽  
Endemic Area ◽  
Protein Profile ◽  
Pathogen Transmission ◽  
Dot Blot ◽  
Protein Extract ◽  
Salivary Gland Extract ◽  
Anopheles Mosquitoes ◽  
Sds Page ◽  
Human Sera

Although malaria had ever been virtually eradicated from Indonesia but currently malaria is recognized as a serious re-emerging threat to public health. This disease is caused by malaria parasite which is transmitted to human host by Anopheles mosquitoes as main vector. It has been widely observed that saliva of mosquito that transmits disease contains several factors that could enhance pathogen infection. Therefore, it should be possible to control pathogen transmission by vaccinating the host against the molecule(s) in saliva that potentiate the infection. However, immunogenic specific component in mosquitoes vectors of Malaria has not yet been identified so far. The objective of this study are to analyze protein profile of SDS-PAGE and to know the immunogity the protein extract of salivary gland from potential vector of Malaria i.e. An. aconitus We used immunogenic reaction between salivary gland extract of these vectors against pool of human sera which were collected from endemic area. The reaction conducted by the dot-blot analyze. SDS-PAGE studies showed 15 major polypeptide bands of 284, 100, 84, 75, 66, 57, 53, 48, 45, 38, 33, 29, 15, 14, and 11 kDa. The dot-blot studies showed that the protein extract of salivary gland from An. aconitus are immunogenic.

scholarly journals IMMUNOGENESITY OF SPESIFIC PROTEIN MOLECULAR WEIGHT 16 KDa (PS16) LEAF OF SIAM CITRUS INFECTED BY CITRUS VEIN PHLOEM DEGENERATION (CVPD) DISEASE

2007 ◽  
Vol 13 (1) ◽  
pp. 63-67
Author(s):  
Made Sritamin ◽  
Aulanni ‘am Aulanni ‘am ◽  
IGP. Wirawan ◽  
Liliek Sulistyowati
Keyword(s):  
Specific Protein ◽  
Disease Spread ◽  
Cellulose Membrane ◽  
Dot Blot ◽  
Citrus Plant ◽  
Rabbit Igg ◽  
Sds Page ◽  
Protein Molecules ◽  
Citrus Production ◽  
Induce Antibody

Citrus Vein Phloem degeneration (CVPD) is an important citrus disese, which damaged citrus plantation and causing decrease of citrus production. In Indonesia, the CVPD disease caused by Liberobacter asiaticum bactery and the disease spread out by vectir insect Diaphorina citri and using infected bud in wood grafting. In infected citrus plant, two specific protein molecules with molecular weigt 16 kDa and 66 kDa are found. These protein molecules are not found in healthy citrus plant. The immunogenicity of PS16 accumulated on leaf of citrus plant infected by CVPD is known yet. The research material were leaves of citrus plant infected CVPD, leaves of healthy citrus plant and reagent used these research are for isolation of the total protein leaf of citrus plant, SDS-PAGE electroforesis, electroelution of PS16, ELISA Methods, Dot-Blot Method, anti-PS16 as aprimery antibody and secondary antibody is anti-Rabbit IgG Conjugated AP. The result of the research showed that of PS16 accumulated on leaf of citrus plant infected CVPD has immunogenic character. It is indicated by increase of the titer anti-PS16 after first immunization ang 2nd booster by indirect ELISA method and can be used to induce antibody (anti-PS16) and so showed that positive reaction between PS16 with anti-PS16. It is indicated by purples dark blue on cellulose membrane by Dot Blot method.

scholarly journals PURIFIKASI PROTEIN IMUNOGENIK 31 DAN 56 kDa DARI KELENJAR SALIVA Aedes aegypti

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Syubbanul Wathon ◽  
Fitria Mutiah ◽  
Rike Oktarianti ◽  
Kartika Senjarini
Keyword(s):  
Salivary Gland ◽  
Aedes Aegypti ◽  
Positive Control ◽  
Protein Fractions ◽  
Immunogenic Protein ◽  
Dot Blot ◽  
Molecular Weights ◽  
Sds Page ◽  
Salivary Gland Protein ◽  
Immunogenic Proteins

Purification of 31 and 56 kDa Immunogenic Proteins from the Salivary Glands of Aedes aegyptiThe salivary gland of arthropod vector contains various bioactive compounds and plays a role in the transmission of pathogens to the host. The host develops anti-salivary antibodies against vector saliva exposure. Our previous research has identified two immunogenic proteins with molecular weights of 31 and 56 kDa from the Aedes aegypti salivary gland protein extract. However, the role of the 31 and 56 kDa immunogenic proteins from saliva Ae. aegypti is not fully known, so it is necessary to purify two immunogenic protein fractions to better specify the target of developing a dengue vaccine. This study aimed to purify the 31 and 56 kDa immunogenic protein fractions by electroelution and dialysis methods. The purification of the two protein fractions has been successful which were confirmed by the SDS-PAGE by the existence of single-band parallel to the positive control. These results were further supported by the dot blot analysis which showed a positive reaction in the form of dark spots in the two protein fractions which were reacted with dengue patients' serum, endemic healthy people, and neonates. These results indicated that the purified 31 and 56 kDa immunogenic protein fraction can be identified by specific antibodies.Keywords: dialysis, electroelution, immunogenic, purification, saliva  ABSTRAKKelenjar saliva vektor arthropoda mengandung berbagai senyawa bioaktif dan berperan dalam transmisi patogen ke tubuh inang. Tubuh inang mengembangkan antibodi anti-saliva terhadap paparan saliva vektor. Penelitian kami sebelumnya telah mengidentifikasi dua protein imunogenik dengan berat molekul 31 dan 56 kDa dari ekstrak protein kelenjar saliva Aedes aegypti. Namun demikian, peranan protein imunogenik 31 dan 56 kDa dari saliva Ae. aegypti belum diketahui sepenuhnya sehingga perlu dilakukan purifikasi dua fraksi protein imunogenik untuk lebih menspesifikkan target pengembangan vaksin dengue. Tujuan penelitian ini untuk melakukan purifikasi fraksi protein imunogenik 31 dan 56 kDa melalui metode elektroelusi dan dialisis. Keberhasilan purifikasi dua fraksi protein 31 dan 56 kDa terbukti dari hasil konfirmasi SDS-PAGE dengan terbentuknya pita tunggal sejajar dengan kontrol positif. Hasil tersebut diperkuat dengan analisis dot blot yang menunjukkan reaksi positif dengan munculnya noktah gelap pada dua fraksi protein tersebut ketika direaksikan dengan serum pasien DBD, penduduk sehat endemik dan neonatus. Hasil ini mengindikasikan bahwa fraksi protein imunogenik 31 dan 56 kDa hasil purifikasi dapat dikenali oleh antibodi spesifik.

G protein-coupled receptors mediate coronary flow- and agonist-induced responses via lectin-oligosaccharide interactions

2014 ◽  
Vol 306 (5) ◽  
pp. H699-H708 ◽  
Author(s):  
Sandra Perez-Aguilar ◽  
David Torres-Tirado ◽  
Guadalupe Martell-Gallegos ◽  
Jimena Velarde-Salcedo ◽  
Ana Paulina Barba-de la Rosa ◽  
...  
Keyword(s):  
G Protein ◽  
Insulin Receptors ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Dot Blot ◽  
Flow Sensing ◽  
Endothelial Surface ◽  
Sds Page ◽  
L Proteins ◽  
G Protein Coupled

Blood flow acts parallel to the coronary luminal endothelial surface layer (LESL) and modulates multiple parenchymal functions via the release of paracrine agents. Evidence indicates that the LESL may be a flow-sensing organelle and that perhaps through flow-induced lectin (L)·oligosaccharide (O) complex formation (L·O) participates in this process. LESL integrins and selectins are both lectinic and flow sensitive, but the L properties of flow-sensitive G protein-coupled receptors (GPCRs) are unknown. Therefore, we investigated the presence of L in the LESL and hypothesized that if flow-sensitive GPCRs are L, flow and O will determine their response to receptor activation. The LESL protein fraction isolated from guinea pig hearts was passed through an affinity chromatography column made of three sugars, mannose, galactose, and N-acetylglucosamine, and the lectinic fraction was eluted. Immune dot blot was used to identify L proteins in the LESL fraction. Our results indicate the following. 1) Two-dimensional SDS-PAGE (2D-SDS-PAGE) of the LESL lectinic fraction revealed at least 167 Ls. 2) Among these Ls, we identified three selectins and the GPCRs: angiotensin II, bradykinin (B2-R), adenosine A1 and A2, prolactin, endothelin, α1-adrenergic (α1A-R), thromboxane A2, β1-adrenergic, β3-adrenergic, and insulin receptors; the first six GPCRs are known to be flow sensitive. 3) The amplitude of receptor-induced vascular responses by α1A-R and B2-R activation (phenylephrine or bradykinin, respectively) was a function of flow and O (hyaluronidate). Our results support a novel mechanism of GPCR-mediated responses to flow via L·O interaction.

scholarly journals Plantaricin IIA-1A5 from Lactobacillus plantarum IIA-1A5 displays bactericidal activity against Staphylococcus aureus

2015 ◽  
Vol 6 (4) ◽  
pp. 603-613 ◽  
Author(s):  
I. Isnafia Arief ◽  
C. Budiman ◽  
B. Sri Laksmi Jenie ◽  
E. Andreas ◽  
A. Yuneni
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Membrane ◽  
Lactobacillus Plantarum ◽  
Histidine Kinase ◽  
Mode Of Action ◽  
Apparent Size ◽  
Gene Encoding ◽  
Disulphide Bonds ◽  
Sds Page ◽  
Quorum Sensor

Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5 isolated from Indonesian beef. This research aimed to identify the genes involved in plantaricin IIA-1A5 production and examine its mode of action against Staphylococcus aureus. It has been reported that a bacteriocin structural gene, plnW, is present in genome of L. plantarum IIA-1A5. Here, we reported the presence of additional genes responsible for plantaricin precursor (plnA and plnEF) and a gene encoding the quorum sensor of histidine kinase (plnB). It indicates that genes involved in production of plantaricin IIA-1A5 are organized in at least two bacteriocin operons (plnABCD, plnEFI) and a structural plnW gene. Purified plantaricin IIA-1A5 yielded a single band in SDS-PAGE with apparent size of 6.4 kDa. Amino acid composition of purified plantaricin IIA-1A5 was mainly composed of cationic glutamic acid and cysteine that allowed the formation of disulphide bonds, suggesting plantaricin IIA-1A5 belongs to the pediocin-subclass of class II bacteriocins. Plantaricin IIA-1A5 displayed remarkable antibacterial activity against S. aureus, which was initiated by the adsorption of plantaricin IIA-1A5 onto the cell membrane of S. aureus. The adsorption is hypothesised to be facilitated by non-ionic interactions as it is reduced by the presence of organic solvents or detergents. This adsorption promoted leakage of cellular metabolites through the cell membrane of S. aureus, as indicated by the release of genetic and proteinaceous material of S. aureus observed at 260 and 280 nm, respectively. The leakage also promoted the release of divalent (Ca2+, Mg2+) and monovalent (K+) cations. The release of these intracellular components might be due to pores formed in the cell membrane of S. aureus by plantaricin IIA-1A5 as shown by scanning electron microscopy. Altogether, the mode of action of plantaricin IIA-1A5 against S. aureus seems to be bactericidal as indicated by lysis of the cell membrane.

scholarly journals Sickle Cell Trait Induces Oxidative Damage on Plasmodium falciparum Proteome at Erythrocyte Stages

2019 ◽  
Vol 20 (22) ◽  
pp. 5769
Author(s):  
Alber Díaz-Castillo ◽  
Neyder Contreras-Puentes ◽  
Ciro Alvear-Sedán ◽  
Carlos Moneriz-Pretell ◽  
Erika Rodríguez-Cavallo ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Clinical Symptoms ◽  
Sickle Cell Trait ◽  
Dot Blot ◽  
Hemoglobin A ◽  
Sds Page ◽  
Erythrocytic Cycle ◽  
Dot Blot Assay ◽  
High Production ◽  
Oxidative Effects

The presence of hemoglobin A-S (HbAS) in erythrocytes has been related to the high production of reactive oxygen species (ROS) and an increased in intracellular oxidative stress that affects the progress of Plasmodium erythrocytic cycle life and attenuates its serious clinical symptoms. Nevertheless, oxidative effects on P. falciparum proteome across the intraerythrocytic cycle in the presence of HbAS traits have not been described yet. Here, an immune dot-blot assay was used to quantify the carbonyl index (C.I) on P. falciparum 3D7 proteome at the different asexual erythrocytic stages. Protein carbonylation on parasites cultivated in erythrocytes from two donors with HbAS increased 5.34 ± 1.42 folds at the ring stage compared to control grown in hemoglobin A-A (HbAA) red blood cells. Whereas at trophozoites and schizonts stages were augmented 2.80 ± 0.52 and 3.05 ± 0.75 folds, respectively. Besides proteins involved in processes of the stress response, recognition and invasion were identified from schizonts carbonylated bands by combining SDS-PAGE with MALDI-TOF-TOF analysis. Our results reinforce the hypothesis that such oxidative modifications do not appear to happen randomly, and the sickle cell trait affects mainly a small fraction of parasite proteins particularly sensitive to ROS.

scholarly journals Design, synthesis and determination of physical and chemical characteristics of glycoconjugates as model for oligosaccharide vaccines against vibrio cholerae

2003 ◽  
Vol 48 ◽  
pp. 15-20
Author(s):  
Aleksandra Grozdanova ◽  
Ana Poceva ◽  
Katerina Milenkova ◽  
Ljubica Suturkova
Keyword(s):  
Vibrio Cholerae ◽  
Cholera Toxin B Subunit ◽  
Dot Blot ◽  
Design Synthesis ◽  
B Subunit ◽  
Safe Level ◽  
Protein Carriers ◽  
Sds Page ◽  
Vibrio Cholerae O1 ◽  
Physical And Chemical

Cholera is toxin-mediated enteroinfection, with epidemic character and there are approximately 120000 death cases per year worldwide. Protection against cholera has not been accomplished due to deficiencies in the licensed vaccines. Serum vibriocidal activity mediated by LPS antibodies is the only immune segment correlated with the resistance of cholera. On the basis of literature data (Robbins JB, 1990; Ogawa Y, 1996) we synthesized glucoconjugates, composed of detoxified LPS from Vibrio cholerae and protein carriers. Conjugate vaccines were prepared by binding acetic acid and hydrazine-treated lipopolysaccharide (LPS) from Vibrio cholerae O1, serotype Inaba, to cholera toxin B-subunit (CT-B) and bovine serum albumin (cBSA). Adipic acid dihydrazide was used for derivatization of oligosaccharides and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as conjugating agent. SDS-PAGE, glycoprotein detection and TLC dot-blot were used for physical and chemical analysis of the prepared four types of conjugates. Safe level of endotoxins, measured by LAL assay was detected in all conjugates. The synthesized conjugates can be used for monitoring immunization schemes on experimental animals. It is to be expected that conjugated vaccines are safe and efficient and that will have high immunogenic and T-dependant characteristics with long immune protection against cholera.

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