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Proteomic studies usually begin with characterizing protein profile, like the molecular weight of protein, which can be done by the SDS-PAGE technique followed by Western Blot. These methods need a standard protein called molecular weight marker (MWM). In this review, the important, basic aspects of either recombinant or native MWM production was discussed, including the type and effect of dyes can be used for preparing a prestained MWM. Moreover, buffer and polyols used in the formulation can also affect the quality and stability. Also, another adjuvant may be needed to increase the robustness of MWM to lower the risk of protease contamination that can breakdown the protein inside the MWM during storage. Understanding those critical aspects will help to produce/formulate a good quality of MWM.

Background Many cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed. However, no size-specific permeability assay suitable for both cell types exists. Results We report the use of intrinsically biotinylated cell components as the target for reporter molecules for assessing permeabilisation. Due to its well-described biotin binding activity, we developed an assay using Streptavidin (SAv) as a molecular weight marker for assessing eukaryotic and prokaryotic cell internalisation, using flow cytometry as a readout. This concept was tested here as part of the development of host DNA depletion strategies for microbiome analysis of formalin-fixed (FF) samples. Host depletion (HD) strategies require differential cell permeabilisation, where mammalian cells but not bacterial cells are permeabilised, and are subsequently treated with a nuclease. Here, the internalisation of a SAv-conjugate was used as a reference for nucleases of similar dimensions. With this assay, it was possible to demonstrate that formalin fixation does not generate pores which allow the introduction of 60 KDa molecules in mammalian or bacterial membranes/envelopes. Among surfactants tested, Saponin derived from Quillaja bark showed the best selectivity for mammalian cell permeabilisation, which, when coupled with Benzonase nuclease, provided the best results for host DNA depletion, representing a new HD strategy for formalin fixed samples. Conclusion The assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.

Alkaline agarose gels are run at high pH, which causes each thymine and guanine residue to lose a proton and thus prevents the formation of hydrogen bonds with their adenine and cytosine partners. The denatured DNA is maintained in a single-stranded state and migrates through an alkaline agarose gel as a function of its size. Other denaturants such as formamide and urea do not work well because they cause the agarose to become rubbery.

The protein purity is generally checked using SDS‐PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS‐PAGE: that is, imaged with Stain‐Free™ technology. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10‐12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 28°C for 12 hr. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Images of the SDS‐PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. No brightness and contrast adjustment was applied. The production of the nanobody CH10‐12 was obtained through a fed‐batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS‐PAGE gel images., This article proposes a protocol for image analysis of electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers in SDS‐PAGE gels as protein standards. The periplasmic production of the nanobody CH10‐12 was obtained through fed‐batch strategy and quantified using the band of 50 kDa in the marker as a reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS‐PAGE gel images.

Slab gel electrophoresis (SGE) is very common tool for DNA, RNA and protein analysis, but it is tedious, labor-intensive, skill-dependent, and relatively slow. Herein, we developed a biochip based SGE system, which can resolve the DNA fragments and record their separation process. By electrophoresis of 50 bp DNA ladder, we found that the 16 DNA fragments were effectively resolved within 14 min. In order to validate its feasibility and practicability, we take periodontal pathogens (e.g., Porphyromonas gingivalis (P.g), Tannerela forsythia (T.f), and Treponema denticola (T.d) ) as an example by separating their polymerase chain reaction products. Experiments demonstrated that P.g , T.d and T.f were diagnosed within 12 min, and the electrophoresis of P.g showed that the detection limit of this system was about 6.4 ng/μl. Such a low cost system is easy to operate, and can effectively improve SGE in the biological experiment, especially for the labs in the third world countries.

This protocol describes steps for the preparation and running of agarose gels and for staining and visualization of DNA in gels using three dyes: ethidium bromide, SYBR Gold, and SYBR Green 1.

A voltage-programming-based capillary gel electrophoresis method with a laser-induced fluorescence detector was developed for the fast and highly sensitive detection of DNA molecules related to angiotensin-converting enzyme insertion/deletion polymorphism, which has been reported to influence predisposition to various diseases such as cardiovascular disease, high blood pressure, myocardial infarction, and Alzheimer's disease. Various voltage programs were investigated for fast detection of specific DNA molecules of angiotensin-converting enzyme insertion/deletion polymorphism as a function of migration time and separation efficiency to establish the effect of voltage strength to resolution. Finally, the amplified products of the angiotensin-converting enzyme insertion/deletion polymorphism (190 and 490 bp DNA) were analyzed in 3.2 min without losing resolution under optimum voltage programming conditions, which were at least 75 times faster than conventional slab gel electrophoresis. In addition, the capillary gel electrophoresis method also successfully applied to the analysis of real human blood samples, although no polymorphism genes were detected by slab gel electrophoresis. Consequently, the developed voltage-programming capillary gel electrophoresis method with laser-induced fluorescence detection is an effective, rapid analysis technique for highly sensitive detection of disease-related specific DNA molecules.

Accurate and reproducible measurement of blood-brain barrier (BBB) integrity is critical in the assessment of the pathophysiology of the central nervous system disorders and in monitoring therapeutic effects. The widely-used low molecular weight marker [(14)C]sucrose is non-specific in the absence of chromatographic separation. The purpose of this study was to develop and validate a sensitive and reproducible LC-MS/MS method for the analysis of stable isotope-modified [(13)C12]sucrose in brain, plasma, and blood to determine BBB permeability to sucrose. After addition of internal standard (IS, [(13)C6]sucrose), the marker and IS were recovered from diluted rat blood, plasma, and brain homogenate by protein precipitation using acetonitrile. The recovery of the marker and IS was almost quantitative (90-106%) for all three matrices. The recovered samples were directly injected into an isocratic UPLC system with a run time of 6 min. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r(2)≥0.99) in the concentration ranges tested for the diluted blood and plasma (10-1000 ng/mL) and brain homogenate (1-200 ng/mL). The lower limit of quantitation of the assay was 0.5 pg injected on column. The assay was validated (n=5) based on acceptable intra- and inter-run accuracy and precision values. The method was successfully used for the measurement of serial blood and plasma and terminal brain concentrations of [(13)C12]sucrose after a single intravenous dose (10 mg/kg) of the marker to rats. As expected, the apparent brain uptake clearance values of [(13)C12]sucrose were low in healthy rats. The method may be useful for determination of the BBB integrity in animal models.

Here we report a new approach for studying protein oligomerization in cells using a single electrophoresis gel. We combined the use of a crosslinking reagent for sample preparation, such as glutaraldehyde, with the analysis of oligomers by Tris-acetate polyacrylamide gel electrophoresis. The use of a 3-15% Tris-acetate polyacrylamide gradient gel allows for the simultaneous analysis of proteins of masses ranging from 10 to 500 kDa. We showed the usefulness of this method for analyzing endogenous p53 oligomerization with high resolution and sensitivity in human cells. Oligomerization analysis was dependent on the crosslinker concentration used. We also showed that this method could be used to study the regulation of oligomerization. In all experiments, Tris-acetate polyacrylamide gel electrophoresis proved to be a robust, manageable, and cost- and time-efficient method that provided excellent results using a single gel. This approach can be easily extrapolated to the study of other oligomers. All of these features make this method a highly useful tool for the analysis of protein oligomerization.

Aim: The present study was undertaken to standardize a convenient method for isolation and purification of β-lactoglobulin (β-lg) from cow milk keeping its antigenicity intact, so that the purified β-lg can be used for detection of cow milk protein intolerance (CMPI). Materials and Methods: Raw milk was collected from Gir breed of cattle reared in Haringhata Farm, West Bengal. Milk was then converted to skimmed milk by removing fat globules and casein protein was removed by acidification to pH 4.6 by adding 3 M HCl. β-lg was isolated by gel filtration chromatography using Sephacryl S-200 from the supernatant whey protein fraction. Further, β-lg was purified by anion-exchange chromatography in diethylaminoethyl-sepharose. Molecular weight of the purified cattle β-lg was determined by 15 percent one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was analyzed by gel documentation system using standard molecular weight marker. Results: The molecular weight of the purified cattle β-lg was detected as 17.44 kDa. The isolated β-lg was almost in pure form as the molecular weight of purified β-lg monomer is 18kDa. Conclusion: The study revealed a simple and suitable method for isolation of β-lg from whey protein in pure form which may be used for detection of CMPI.

Visualizing nucleic acids by gel electrophoresis is one of the most common techniques in molecular biology, and reference molecular weight markers or ladders are commonly used for size estimation. We have created the pPSU1 & pPSU2 pair of molecular weight marker plasmids which produce both 100 bp and 1 kb DNA ladders when digested with two common restriction enzymes. The 100 bp ladder fragments have been optimized to migrate appropriately on both agarose and native polyacrylamide, unlike many currently available DNA ladders. Sufficient plasmid DNA can be isolated from 100 ml E. coli cultures for the two plasmids to produce 100 bp or 1 kb ladders for 1000 gels. As such, the pPSU1 and pPSU2 plasmids provide reference fragments from 50 to 10000 bp at a fraction of the cost of commercial DNA ladders. The pPSU1 and pPSU2 plasmids are available without licensing restrictions to nonprofit academic users, affording freely available high-quality, low-cost molecular weight standards for molecular biology applications.

This unit includes the necessary information to conduct neutral/neutral and neutral/alkaline two-dimensional and neutral/neutral/alkaline three-dimensional agarose gel electrophoresis. The methodology has been optimized over the years to gain a better outcome from the hard-to-interpret signals of human papilloma virus replication intermediates obtained from two- and three-dimensional agarose gels. Examples of typical results and interpretation of replication intermediate patterns are included, and the outcomes of multiple-dimension assays are assessed using previously published experimental data. © 2017 by John Wiley & Sons, Inc.

Objective The performance of comparative analysis of sensitivity and results of detection of avian influenza virus by real time polymerase chain reaction (PCR-RT) and loop-mediated isothermal amplification of the nucleic acids (LAMP) was the main goal of the study. Introduction As part of this surveillance study for Avian Influenza both active and passive surveillance samples were tested using PCR and also utilized to validate the LAMP method. Active surveillance samples include pathological material and tracheal and cloacal swabs from ill poultry, which were subsequently assessed for avian influenza during diagnosis, and birds collected by hunters. Passive surveillance included environmental samples such as sand and bird faeces. Active surveillance samples were taken mostly from poultry farms across Ukraine, where infected birds are required to be diagnosed by State Scientific Research Institute of Laboratory Diagnostics and Veterinary Sanitary Expertise (SSRILDVSE) by Ukraine Law. Passive surveillance samples were taken primarily during the annual bird migration season. Development of simple, sensitive, and cheap methods for diagnostics of avian influenza is a very important task for practical veterinary medicine. LAMP is one of such methods. The technique is based on isothermal amplification of nucleic acids. It does not require special conditions and equipment (PCR cyclers), therefore it is cheaper in comparison with PCR. Accurate diagnosis is necessary for determining the risk associated with avian influenza in Ukraine and along the Dnipro River during the migratory season. Methods For the research, we used PCR-RT commercial kit Bird-Flu-PCR (Ukrzoovetprompostach, Ukraine), LAMP (the protocol has been optimized and patented by SSRILDVSE), QIAamp ® Viral RNA Mini Kit. For the study, we used pathological and biological materials from birds, which were sent to the SSRILDVSE from all regions of Ukraine according to the 2013–2014 State monitoring plan. Set up of the real time PCR reactions and parameters of amplifications are indicated in the instruction to the kit. The following protocol was used to set up the RT- LAMP: 2.5 μ L 10 X Thermopol buffer, 1 mmol/L betaine, 5 mmol/L MgSO4, 1.4 mmol/L - BNTP, 12.5 μ mol/L SYBR GREEN, 0.5 mmol/L MnCL2, up to 25 μ L Nuclease-free water, 8 U Bsm DNA polymerase, 0.1 μ M/1 of F3, 0.1 μ M/1 of B3, 0.8 μ M/1 of FIP, 0.8 μ M/1 of BIP, 0.4 μ M/1 of LF, 0.4 of LB, 2 μ L cDNA. During our work, we used the following optimal temperature and time for the amplification – 59°C and 60 minutes. The sensitivity of diagnostic kit Bird-Flu-PCR and RT- LAMP was determined by testing cDNA of the reference strain of AIV H5N1, which was provided to us by NSC Institute for Experimental and Clinical Veterinary Medicine (Kharkiv, Ukraine). For the standard, we employed concentration in the range of 10.0-0.01 ng/sample. Results Table 1. This table shows the reproducibility results obtained by both methods. However, taken into account absence of highly pathogenic avian influenza virus circulating in Ukraine during the studied period, it was not possible to confirm these results with protocols of positive samples. Table 2. It has been established that the sensitivity of PCR-RT kit Bird-Flu- PCR is 0.01 ng/sample for gene M and 0.1 ng/sample for subtype H5N1. Fig. 1. Visual detection of LAMP products with different concentrations of cDNA of avian influenza virus (ng per sample): 1 – 10; 2 – 5; 3 – 1.0; 4 – 0.1; 5–7 – 0.01; 8–9 – 0.1; 10 – negative. We have examined the LAMP results using electrophoresis for the confirmation of visual detection and correct interpretation of the results (Fig. 2). Fig.2. Electrophoresis results for LAMP products. M – molecular weight marker; 1 – 10.0; 2 – 5.0; 3 – 1.0; 4 – 0.1; 5–7 – 0.01; 8 - negative control . It has been established that the sensitivity of LAMP is 0.1 ng/sample. Slightly lower sensitivity of LAMP in comparison to PCR-RT can be explained by visual detection of the products of the LAMP reaction. Conclusions 1. Sensitivity of both methods is high. 2. LAMP is a perspective screening method for the diagnosis of viral infectious diseases supported by confirmation of positive results by PCR-RT.

Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate binding modules (CBMs). In recent years, carbohydrate surface binding sites of proteins mostly enzymes have also been investigated by this method. Here, we describe a protocol for identifying binding interactions between enzyme catalytic modules and a variety of carbohydrate ligands.

Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate and EDTA (TAE) or Tris, borate and EDTA (TBE). Gels are run at a low, constant voltage (~ 10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent upon the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20–25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10 – 100 bp): high voltages and short run times produced sharper bands and higher resolution.

Objective: To explore a fast and sensitive method for screening T cell receptor (TCR) biases through comparison of silver staining for polyacrylamide gel electrophoresis and ethidium bromide staining for agarose gel electrophoresis. Methods: The lymphocytes were isolated from peripheral blood which collected from ten patients with T1DM; the total RNA was extracted followed by transformed to cDNA, and then electrophoresis was carried out on polyacrylamide and agarose gel simultaneously, finally stained with silver and ethidium bromide dying respectively. Results: The pictures of TCR Vβ biases detected with silver staining for polyacrylamide gel electrophoresis were very similar to that with ethidium brominde staining for agarose gel electrophoresis, Vβ7, Vβ11 and Vβ17 were the mainly biased genes with higher frequencies, 10 and Vβ13.2 were the common genes which did not biased in all the ten patients. In some Vβ genes, the number of the bands detected with the former technique was more than that with the latter methods. So did the clarity of some bands. Conclusions: Silver staining for polyacrylamide gel electrophoresis is a fast, sensitive and safe method for screening the biases of TCR Vβ genes.

The present study characterizes the effects of lysophosphatidic acid (LPA) on blood–brain barrier (BBB) permeability focusing specifically on the time of onset, duration, and magnitude of LPA-induced changes in cerebrovascular permeability in the mouse using both magnetic resonance imaging (MRI) and near infrared fluorescence imaging (NIFR). Furthermore, potential application of LPA for enhanced drug delivery to the brain was also examined by measuring the brain accumulation of radiolabeled methotrexate. Exposure of primary cultured brain microvessel endothelial cells (BMECs) to LPA produced concentration-dependent increases in permeability that were completely abolished by clostridium toxin B. Administration of LPA disrupted BBB integrity and enhanced the permeability of small molecular weight marker gadolinium diethylenetriaminepentaacetate (Gd-DTPA) contrast agent, the large molecular weight permeability marker, IRdye800cwPEG, and the P-glycoprotein efflux transporter probe, Rhodamine 800 (R800). The increase in BBB permeability occurred within 3 minutes after LPA injection and barrier integrity was restored within 20 minutes. A decreased response to LPA on large macromolecule BBB permeability was observed after repeated administration. The administration of LPA also resulted in 20-fold enhancement of radiolabeled methotrexate in the brain. These studies indicate that administration of LPA in combination with therapeutic agents may increase drug delivery to the brain.

Topology plays a key role in the biophysics of DNA, and is intimately related to its functioning. For instance, transcription of a gene redistributes twist locally to create what is known as supercoiling, while catenanes or knots can prevent cell division, hence they need to be quickly and accurately removed by specialised enzymes known as topoisomerases. But how can one establish experimentally the topological state of a given DNA molecule? By far the most successful and widely used technique for this is gel electrophoresis (Calladine et al. 1997; Bates and Maxwell 2005). This method exploits the empirical observation that the mobility of a charged DNA molecule under an electric field and moving through a gel depends on its size, shape and topology (Bates and Maxwell 2005; Stasiak et al. 1996). Nowadays, gel electrophoresis is a ubiquitous technique (Calladine et al. 1997; Viovy 2000; Dorfman 2010), since it readily allows the separation of polymers with different physical properties and it is systematically used for DNA identification and purification (Calladine et al. 1997).

Polyacrylamide gel electrophoresis (PAGE) constitutes a powerful technique for the efficient purification of RNA molecules dedicated to applications that require high purity levels. PAGE allows for the fractionation of RNA obtained from cell extracts, chemical or enzymatic synthesis, or modification experiments. Native or denaturing conditions can be chosen for analytical or preparative-scale separations and the nucleotide resolution can be tuned by changing the percentage and reticulation of the gel material. In this protocol, we focus on the preparation of milligram-scale amounts of ~200 nucleotides (nt) RNA molecules that were used in subsequent crystallization experiments.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the most important and widely used technology which is mainly used to analyze the protein molecular weight. Fibrin zymography based on the SDS-PAGE is the best method for qualitative analysis of unknown plasminogen activators (PAs), especially for the analysis of molecular weight. In electrophoresis technique, molecular weight marker is the most important factor. However, it is difficult to detect protein molecular weight markers in fibrin zymography. In this study, some important factors, such as concentrations of fibrinogen and plasminogen, are discussed. Our results provide an efficient and convenient method which can clearly exhibit the dark blue bands of protein molecular weight (MW) markers and the transparent bands of PAs against the light blue background on one gel at the same time, and show high sensitivity.

An S-layer (surface layer) is a part of the cell envelope commonly found in bacteria, as well as among archaea. It consists of a monomolecular layer composed of identical proteins or glycoproteins. As for many bacteria, the S-layer represents the outermost interaction zone with their respective environment, its functions are very diverse and vary from species to species; subject of this study was to find out the prevalence of Nanostructure of Surface layer among the Bacillus cereus isolated from the skin of clinical staff. Relevant information to Surface Layer was extracted from articles that listed in Pubmed, Elsivier Science, and Yahoo from 1995 to 2010 years. The research was performed with laboratory method during 2005-2007 years in Azzahra Hospital and Esfahan University. Bacterial identification was performed with standard bacteriological methods and selective culture medium was applied in preparation of samples. Bacterial samples were cultured in TSA, for 16 hour, in aerobic condition then separated the surface proteins to run electrophoresis with molecular weight marker. S-Layer in Bacillus cereus has 97KD molecular weight. Among the 13 Bacillus cereus strains isolated from skin of staff, 11 (84.60%) showed positive for S-layer. S-layers in pathogenic bacteria offer protection against bacteriophages and phagocytosis, resistance against low pH ,adhesion, stabilisation of the membrane and provide adhesion sites for exoproteins due to more pathogenesis, infection resistant and antibiotic resistant to bacteria. Result of this study shows the prevalence of S-layer in pathogen bacteria and as important to determine S-layer producer strains in laboratory.

Recovery of DNA from agarose gel electrophoresis is a basic operation during molecular cloning. Circular or linear DNA fragments which vary from 1.5 to 6.5 kb and correspond to 1 kb marker can be recovered from 0.8 to 1.0% agarose gel smoothly with a simple and rapid trap method. The recovery efficiency could be more than 70% and the quality of the recovered DNA is proved to be good enough for future research. Here, we provided another good method to recover DNA from agarose gel besides using commercial kits. Key words: Trap method, recovery of DNA, agarose gel electrophoresis.

Zymography detects and characterizes proteolytic enzymes by electrophoresis of protease-containing samples into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing a copolymerized protein substrate. The usefulness of zymography for molecular weight determination and proteomic analysis is hampered by the fact that some proteases exhibit slower migration through a gel that contains substrate protein. This article introduces electrophoretic transfer protein zymography as one solution to this problem. In this technique, samples containing proteolytic enzymes are first resolved in nonreducing SDS-PAGE on a gel without protein substrate. The proteins in the resolving gel are then electrophoretically transferred to a receiving gel previously prepared with a copolymerized protein substrate. The receiving gel is then developed as a zymogram to visualize clear or lightly stained bands in a dark background. Band intensities are linearly related to the amount of protease, extending the usefulness of the technique so long as conditions for transfer and development of the zymogram are kept constant. Conditions of transfer, such as the pore sizes of resolving and receiving gels and the transfer time relative to the molecular weight of the protease, are explored.

Detection methods for immunoblot analysis are often based on peroxidase conjugates. However, molecular weight markers directly detectable for general use in such systems are not available. Here, we describe the preparation of a direct molecular weight marker consisting of heme-tagged proteins, whose enzymatic activities make them detectable simultaneously with the antigen in peroxidase-based immunoblot systems. The peroxidase activity results from the covalent attachment of heme to selected engineered periplasmic proteins, catalyzed by the cytochrome c maturation system of Escherichia coli. The newly designed heme-tagged proteins were combined with a previously constructed heme-tagged maltose-binding protein and cytochrome c. The resulting heme ladder was shown to be suitable as a protein standard for direct molecular weight estimation in immunoblot analysis due to the peroxidase activity of its constituents. The heme ladder consists of proteins between 12 and 85 kDa and can be produced at low cost. The marker was stable when kept at 4, −20, and −80 °C for >6 months.

The application of pore-gradient polyacrylamide gel electrophoresis (PG-PAGE) incorporated with carbon nanotube modified by Triton X-100 and carboxylation so as to improve the separation of human serum proteins is reported. The novel PG-PAGE was made by adding water-soluble single-walled carbon nanotubes (CNTs) when preparing the polyacrylamide gel. Significant improvements in separation of complement C3 protein and haptoglobin (Hp) in human serum were achieved. It was estimated that the interactions between the hydrophilic groups on the proteins and the surface of the CNTs result in different adsorption kinetics of complement C3 and Hp subtype on the nanoparticles incorporated in the gel, thus enhancing the separation of the two proteins in serum. This new CNT matrix-assisted PG-PAGE method for enhanced separation of complement C3 and Hp in human serum was successfully applied to distinguish the samples from liverish patients and healthy people.

One of the fundamental challenges in studying biomacromolecules (e.g. nucleic acids and proteins) and their complexes in a biological system is isolating them in their structurally and functionally intact forms. Electrophoresis offers convenient and efficient separation and analysis of biomacromolecules but recovery of separated biomacromolecules is a significant challenge. In this study, DNAs of various sizes were separated by electrophoresis in an acid-degradable polyacrylamide gel. Almost 100% of the nucleic acids were recovered after the identified gel bands were hydrolyzed under a mildly acidic condition and purified using anion exchange resin. Further concentration by centrifugal filtration and a second purification using ion exchange column chromatography yielded 44-84% of DNA. The second conventional (non-degradable) gel electrophoresis confirmed that the nucleic acids recovered from acid-degradable gel bands preserved their electrophoretic properties through acidic gel hydrolysis, purification, and concentration processes. The plasmid DNA recovered from acid-degradable gel transfected cells significantly more efficiently than the starting plasmid DNA (i.e. improved biological activity via acid-degradable PAGE). Separation of other types of nucleic acids such as small interfering RNA using this convenient and efficient technique was also demonstrated.

A method for the purification of 20S proteasome (20S core particle, CP) was developed by combining differential centrifugations with nondenaturing polyacrylamide gel electrophoresis (native-PAGE), irrespective of species origins of CPs. CP purified from human erythrocytes was subjected to proteomic analysis by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), revealing 33 spots of subunit isoforms with different molecular weights and isoelectric points. Furthermore, other four CPs were purified from yeast, mouse liver, and two pancreatic cancer cell lines SW1990 and PANC-1 using the method mentioned above and subjected to proteasome heterogeneity analysis by two-dimensional native/SDS-PAGE (native/sodium dodecyl sulphate polyacrylamide gel electrophoresis), in combination with CP from erythrocytes. In conclusion, the method described acts as a rapid and effective tool for CP isolations, and the results obtained may serve as a footstone for the investigations of species-dependent proteasome heterogeneity.

This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are primarily due to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 +/- 0.01) x 10(-4) cm2/V s in 40 mM Tris-acetate-EDTA buffer at 20 degrees C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration.

Proteins labeled with a luminescent lanthanide chelate, {{2,2',2'',2'''-{4'-{[(4,6-dichloro-1,3,5-triazin-2-yl)amino]biphenyl-4-yl}-2,2':6',2''-terpyridine-6,6''-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III) (DTBTA-Eu(3+)),were analyzed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) slab gel electrophoresis and hydroxyethyl cellurose gel capillary electrophoresis with a time-resolved fluorometric detector. The metal ion of the luminescent lanthanide chelate did not dissociate from the ligand during electrophoresis, and the luminescence was retained. On a slab gel, the band of DTBTA-Eu(3+)-labeled streptavidin was apparently less broad than that of AlexaFluor 488-labeled streptavidin. DTBTA-Eu(3+) in SDS-PAGE slab gel is stable, and the gel after electrophoresis can be dried and stored for at least one year without luminescence fading. In capillary gel electrophoresis (CGE), five labeled proteins with different molecular weight were separated, and a good correlation was observed between the molecular weight and the migration time. Although the detection limits of these proteins determined in buffer solutions of the capillary electrophoresis were not better than those reported for CGE with laser-induced-detection, the detection limits of the same proteins in the present CGE system were not significantly deteriorated in serum solutions compared to those in buffer solutions, and the advantage of using time-resolved detection has been shown.

DNA molecular weight markers are routinely used in agarose gel electrophoresis. Here we report a method called PCR-synthesized marker (PSM) to generate DNA molecular rulers by PCR in the laboratory. This strategy can also be used to produce 100 bp RNA molecular weight markers by run-off transcription.

Monoclonal antibodies against S-tagged fusion proteins expressed in pET vectors were generated and further characterized. Most pET vectors contain a 15-meric S-tag as a fusion tag for the detection of recombinant proteins. Two antibodies, G18BA3 and G18BE8, recognized this S-tag in enzyme immunoassay and Western blot. Their epitopes were mapped using peptide array technology and were confirmed to be AAKFERQHMDSPD. This corresponds to the C-terminal region of the S-tag plus additional amino acids P and D, which are also present in most available pET vectors. Amino acid substitution analysis revealed several essential residues for binding. The binding motif was therefore FExxHxDxxD for G18BA3 and AxxFExxH for G18BE8. Since some commercially available protein standards are expressed in pET vectors, G18BA3 and G18BE8 were also found to detect the ladder bands of a molecular weight marker on immunoblot analysis. Both antibodies should be highly useful for the simultaneous detection of recombinant pET vector-expressed fusion proteins and protein molecular weight standards in Western blotting, especially when chemoluminescent detection systems are used.

In proteomics, one-dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein fractionation prior to mass spectrometric analysis to enhance the dynamic range of analysis and to improve the identification of low-abundance proteins. Such protein prefractionation works well for quantitation strategies if the proteins are labeled prior to separation. However, because of the poor reproducibility of cutting gel slices, especially when small amounts of samples are analyzed, its application in label-free and peptide-labeling quantitative proteomics methods has been greatly limited. To overcome this limitation, we developed a new strategy in which a DNA ladder is mixed with the protein sample before PAGE separation. After PAGE separation, the DNA ladder is stained to allow for easy, precise, and reproducible gel cutting. To this end, a novel visible DNA-staining method was developed. This staining method is fast, sensitive, and compatible with mass spectrometry. To evaluate the reproducibility of DNA-ladder-assisted gel cutting for quantitative protein fractionation, we used stable isotope labeling with amino acids in cell culture (SILAC). Our results show that the quantitative error associated with fractionation can be minimized using the DNA-assisted fractionation and multiple replicates of gel cutting. In conclusion, 1D PAGE fractionation in combination with DNA ladders can be used for label-free comparative proteomics without compromising quantitation.

For RNA size separation in a small sample volume (

By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K(i)) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs.

A new native protein gel system was recently developed that enables the rapid and convenient analysis of virtually all soluble proteins, in particular including basic proteins, in their native oligomeric states. This gel system combines the addition of negative charges to the proteins by the dye SERVA Blue G with a Tris-histidine discontinuous buffer system and the use of polyacrylamide gradient gels. The use of histidine for sample focusing rather than glycine as a slow dipolar ion following from the cathode buffer serves to improve migration of basic proteins. In this review, the principle of function as well as the advantages and disadvantages of the new gel system are discussed in the context of other native protein gel systems and further methods for the analysis of the oligomeric state of a protein.

Agarose gel electrophoresis, which separates and sizes linear DNA and RNA fragments, is arguably the most basic and essential technique in molecular biology. It is commonly employed for analysis of PCR products, plasmid DNA, and products of restriction enzyme digestion. It is the first step for analysis of specific DNA and RNA fragments by northern and Southern blots. In this unit, we provide both written instructions and photographic images to take the reader from preparing a first agarose gel to analyzing results and determining the size of sample DNA. We include two protocols: agarose gel electrophoresis (commonly used to analyze DNA) and denaturing gel electrophoresis (for analyzing RNA). We have divided each protocol into four basic steps: (1) preparing and pouring the agarose gel; (2) preparing and loading samples; (3) running the agarose gel; and (4) staining the gel using the fluorescent stain ethidium bromide to visualize DNA and RNA. © 2015 by John Wiley & Sons, Inc. Keywords: DNA; RNA; separation; sizing; ethidium bromide; voltage; TAE; TBE; bromphenol blue; xylene cyanol; northern blotting; Southern blotting

Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA fragments.

Genetic diversity of twelve Indian wheat genotypes was assessed on the basis of seed storage protein profiling on 10 and 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Out of twelve genotypes, six genotypes are resistant to salinity, whereas remaining are susceptible. Genetic diversity was calculated using Shannon index, Nei’s index and unweighted pair group method with arithmetic mean (UPGMA) cluster analysis by constructing an electrogram of fragments of proteins, which were used to calculate Jaccard’s similarity coefficients between these varieties. Seed storage protein analyzed by 15 subunits ranging from 14.3-97.4 KDa protein molecular weight marker (PMW-M). The similarity coefficient ranged from 29% to 100%. The detected variation could be used to provide useful information on individual enzymes or transporters, activity as well as modification of structural protein, protein-protein interactions and stress dependent protein movement and can be used to classify adapted cultivars to improve the efficiency of wheat breeding programmes.

Summary Cross-linked homogeneous 5 per cent polyacrylamide gel in rod form is a convenient medium for sharp analytical electrophoretic separation in microgram quantities of most serum proteins and of many biological ampholytes.

Contour-clamped homogeneous electric field (CHEF) electrophoresis is a technique of pulsed-field gel electrophoresis that enables the resolution of large fragments of DNA that cannot be resolved by conventional gel electrophoresis. The procedure involves the application of controlled electric fields that change direction at a predetermined angle to samples of DNA that have been embedded in an agarose gel matrix and digested with a restriction endonuclease. Adjustment of the electrophoresis conditions enables the separation of DNA fragments with lengths from 10 kilobases up to 9 megabases in a size-dependent manner in agarose gels. The banding patterns can be used for epidemiological typing, the separated DNA can be immobilized onto a membrane and used for genetic mapping, or individual fragments can be extracted and used for downstream genetic manipulations. The protocol requires specialized equipment and can be completed in a maximum of 7 days.

Bent DNAs are known to migrate slower than ordinary DNA in non-denaturing polyacrylamide gel electrophoresis. In contrast, several satellite DNAs have been shown to migrate fast. The structural property that causes the fast migration, however, is not clarified so far on molecular basis. We have investigated the structural property of a satellite DNA, which contains consecutive purine sequences and migrates faster in gel, by CD spectroscopy. Partial formation of an A-form-like structure has been suggested. Reduction in DNA length due to the formation of the A-form-like structure may be responsible for the fast migration. The pronounced rigidity of DNA may also contribute to the behavior.

The application of carbon nanotube polyacrylamide gel electrophoresis (PAGE) is being reported so as to improve selectivity of analysis. A novel polyacrylamide gel was manufactured by adding multi-walled carbon nanotubes (MWNT) when producing the polyacrylamide gel. Carbon nanotube-matrix assistance was used to separate apolipoprotein A-I and complement C3 with native polyacrylamide gel electrophoresis. Significant improvement of the relative selectivity was achieved in the separation between apolipoprotein A-I and complement C3, because of the hydrophobic change of the carbon nanotube surface non-covalently functionalized by the surfactant Triton X-100. These two proteins were identified by MS/MS after electrophoresis. The change of the observed mobility can also be noticed using other nanomaterials such as nano-TiO2 and nano-Al2O3, but no significant improvement of relative selectivities could be observed with the latter nanoparticles.

We describe a method to facilitate electrophoretic separation of high-molecular-weight RNA species, such as ribosomal RNAs and their precursors, on agarose-formaldehyde gels. Two alternative "pK-matched" buffer systems were substituted for the traditionally used Mops-based conductive medium. The key advantages include shortened run times, a 5-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting.

Prevention of artifactual protein oxidation occurring during sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis is critical for identifying physiological protein oxidation implicated in human diseases due to the routine use of gel electrophoresis to separate the multiple proteins in proteomic studies. To develop a methodology that completely prevents artifactual protein oxidation in SDS acrylamide gel electrophoresis, cytochrome c was electrophoresed on polyacrylamide gels and subjected to trypsin in-gel digestion followed by tryptic peptide analysis by mass spectrometry. It was found that degassing the acrylamide solution to remove molecular oxygen prior to gel polymerization is a crucial process to protect the electrophoresed protein from reactive oxygen species generated during electrophoresis. However, significant artifactual protein oxidation remains that can only be eliminated entirely, if proteins are electrophoresed on an SDS gel photopolymerized with flavin as the photoinitiator and thioglycolate included in the cathode buffer as a reactive oxygen species scavenger. Using this combination of methodologies, cytochrome c isolated from adult rat heart mitochondria was purified and digested followed by mass spectrometric analysis, demonstrating the requisite high resolution of the polyacrylamide gel and the entire elimination of artifactual oxidation.

Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after γ interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics.

In this paper, we describe a technique to concentrate and separate low-abundant proteins in human plasma. A human plasma sample was fractionated by ammonium sulfate and the precipitate obtained at 35% saturated ammonium sulfate was dialyzed and subjected to isoelectric focusing employing an agarose slab gel (55mm×73mm×3mm, 1% agarose). The IEF gel was cut into strips and each strip was transferred on the top of an SDS-containing column gel (5% T, 5% C polyacrylamide gel) prepared in a Pasture pipette. After SDS electrophoresis, each pipette column gel was transferred onto a top of a micro slab gel (38mm×38mm×1mm, 8-17% T, 5% C polyacrylamide gradient gel) and second SDS gel electrophoresis was run. If the step of pipette electrophoresis was deleted, the proteins appeared as horizontal faint bands on the slab gel. By using the three-step electrophoresis, the low-abundant proteins were detected as densely stained spots on the slab gel with 10-25 fold higher concentration of corresponding proteins than in the case of two-step electrophoresis. The spots on the slab gels after the three-step electrophoresis have been subjected to protein identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The results showed the advantages of the present technique for the structural analysis of low-abundant proteins in human plasma.

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.