Optimizing soluble protein extraction and two-dimensional polyacrylamide gel electrophoresis quality for extremophile ciliates

An efficient protein extraction methodology is quite important for sample preparation and subsequent 2-D PAGE and MS analysis. Cell lysis is the first step in protein extraction and purification. Many techniques are available for cell disruption, including physical and detergent-based methods. Here, we report on a very fast and efficient detergent-free Tris-based method to extract the soluble fraction proteins of extremophile ciliates, comparing it with a detergent-based protocol. This comparison has been carried out by means of 2-D PAGE and subsequent MALDI-compatible silver staining of protein samples obtained from the intensely pigmented hypersaline ciliate Fabrea salina and the Antarctic hypotrich ciliate Euplotes focardii. Our results indicate that this fast and easy extraction method allows to obtain more clear crude extracts and more spot-abundant polyacrylamide gels.     

Quantitative Analysis of Protein–Protein Interactions by Native Page/Fluorimaging

We have developed a new quantitative native PAGE mobility shift assay, which allows for the measurement of binding affinities for interacting protein pairs, one of which is fluorescently labelled. We have used it to examine recognition of the Simian virus 40 (SV40) large tumour T-antigen (T-ag) nuclear localisation sequence (NLS) by members of the importin (Imp) superfamily of nuclear transport proteins. We demonstrate that the T-ag NLS binds to the Imp α/β heterodimer in NLS-dependent manner, determining that it binds with eight-fold higher affinity (340 nM), when compared to Imp α alone, consistent with autoinhibition of Imp αwhen not complexed with Imp β. The mobility shift assay is able to detect nM binding affinities, making it a sensitive and useful tool to analyse protein–protein interactions in solution.     

Development of a rapid and improved two‐dimensional electrophoresis method for the separation of protein lysate

Researchers frequently use two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D‐PAGE method, which ‘inverts’ the dimension of protein separation of the conventional 2D‐PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first‐dimension separation was conducted on a gel‐free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second‐dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D‐PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D‐PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D‐PAGE. Overall, the i2D‐PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.     

Identification of Proteins Using Affinity Affi-Gel Simultaneously with Acrylamide Electrophoresis

Abstract

This work presents a method in which a desired protein may be separated from a biological protein preparation and identified by highly specific monoclonal antibody. The desired protein may be isolated from crude protein preparations by this method. The protein remains intact and stable throughout the procedure. This method utilizes common nonreducing conditions of polyacrylamide gel electrophoresis (PAGE), and Western Blot transfer of protein. This isolation of the desired protein can be accomplished for a microgram or tens of micrograms of material. The application of Western blot technique with monoclonal antibody permits the highest specificity in terms of identifying the target protein. In addition to identifying the target protein the method can isolate sufficient amounts for further characterization.     

Ultrasound assisted three phase partitioning of a fibrinolytic enzyme

The present investigation is aimed at ultrasound assisted three phase partitioning (UATPP) of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Three phase partitioning integrates the concentration and partial purification step of downstream processing of a biomolecule. Three phase system is formed with simultaneous addition of ammonium sulfate to crude broth and followed by t-butanol. UATPP of a fibrinolytic enzyme was studied by varying different process parameters such as ammonium sulfate saturation concentration, pH, broth to t-butanol ratio, temperature, ultrasound frequency, ultrasonication power, and duty cycle. The optimized parameters yielding maximum purity of 16.15-fold of fibrinolytic enzyme with 65% recovery comprised of 80% ammonium sulfate saturation, pH 9, temperature 30 °C, broth to t-butanol ratio 0.5 (v/v), at 25 kHz frequency and 150 W ultrasonication power with 40% duty cycle for 5 min irradiation time. SDS PAGE analysis of partitioned enzyme shows partial purification with a molecular weight in the range of 55–70 kDa. Enhanced mass transfer of UATPP resulted in higher fold purity of fibrinolytic enzyme with reduced time of operation from 1 h to 5 min as compared to conventional TPP. Outcome of our findings highlighted the use of UATPP as an efficient biosepartion technique.     

Subcomplexes of mitochondrial complex V reveal mutations in mitochondrial DNA

Complex V, site of the final step in oxidative phosphorylation, uses the proton gradient across the inner mitochondrial membrane for the production of ATP. It is a multi‐subunit complex composed of a catalytic domain (F₁) and a membrane domain (F₀) linked by two stalks. Subcomplexes of complex V containing the F₁ domain have previously been reported in small series of patients. We report the results in tissue samples and/or cultured skin fibroblasts studied by blue native PAGE followed by activity staining in the gel. Catalytically active subcomplexes of complex V were detected in 66 tissues originating from 53 patients. In 29 of the latter (55%), a mitochondrial DNA (mtDNA) defect was identified. Twelve patients had a pathogenic point mutation in a mitochondrial tRNA, one a large mtDNA deletion, 12 showed mtDNA depletion and four had a mutation in the MT‐ATP6 gene. We conclude that the presence of subcomplexes of complex V is a valuable indicator in the detection of mtDNA defects.     

Sequence determination of αs1‐casein isoforms from donkey by mass spectrometric methods

Four co‐eluting components, with experimentally measured M_r of 23 658, 23 786, 24 278 and 24 406 Da, were detected by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) analysis in the dephosphorylated casein fraction of a milk sample collected at middle lactation stage from an individual donkey belonging to the Ragusano breed. By coupling RP‐HPLC, two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE), enzymatic digestions, MALDI‐TOF MS and capillary RP‐HPLC/nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) analyses, the four components were identified as donkey's α_s1‐CNs and their sequences completely characterized, using the known mare's α_s1‐CN (GenBank Acc. No. AAK83668; M_r 23750.7 Da) as reference. The proteins with M_r of 23 786 and 23 658 Da differ in the presence of a glutamine residue at position 83 in the full‐length component and present the amino acid substitutions Q⁸→H and H¹¹⁵→Y with respect to the mare's α_s1‐CN. The other two components with M_r 24 406 and 24 278 Da, which also differ in the presence of a glutamine residue at position 88 in the full‐length component, show the insertion of the pentapeptide HTPRE between Leu³³ and the Glu³⁴. The two α_s1‐CNs bearing the pentapeptide insertion were named variants A (202 amino acids; M_r 24 406) and A₁ (201 amino acids; M_r 24 278), whereas the two α_s1‐CNs without the pentapeptide were named variants B (197 amino acids; M_r 23 786) and B₁ (196 amino acids; M_r 23 658). Copyright © 2009 John Wiley & Sons, Ltd.     

Albumin‐bound low molecular weight thiols analysis in plasma and carotid plaques by CE

We describe a new method for the quantification of low molecular weight thiols, as homocysteine, cysteine, cysteinylglycine, glutamylcysteine and glutathione bound to human plasma albumin. After albumin isolation and purification by SDS‐PAGE, thiols were freed from protein with tri‐n‐butylphosphine and successively derivatized with 5‐iodoacetamidofluorescein. Samples were then injected and quantified in about 18 min by CE with laser induced fluorescence detection. Precision tests indicate a good repeatability of the method both for migration times (RSD<0.63%) and areas (RSD<2.98%). The method allows to measure all five low molecular weight thiols released from just 3 μg of albumin thus improving the other described methods in which only three or four thiols were detected. Due to the elevated sensitivity (LOD of 0.3 pM for all thiols), also low molecular weight thiols bound to albumin filtered in tissues could be quantified.     

A novel Bicine running buffer system for doubled sodium dodecyl sulfate - polyacrylamide gel electrophoresis of membrane proteins

A novel, Bicine-based SDS-PAGE buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and running buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.