Comparison and optimization of SAR-PAGE tests for erythropoietins in doping analysis.

Erythropoietins (EPOs) are substances listed in S2 of the World Anti-Doping Agency (WADA) Prohibited List and are used commonly by athletes to increase endurance performance. According to the current WADA Technical Documents, sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) followed by western blotting to differentiate erythropoietins based on their molecular weights is the only method that can be used for both screening and confirmation of all types of erythropoietins. The efficiency of immunopurification and protein transfer is crucial for ensuring the selectivity and sensitivity of erythropoietin detection. Several comparisons and optimization of the SAR-PAGE tests were conducted in this study. We optimized the first blotting conditions and then compared different immunopurification methods based on their selectivity, repeatability, and sensitivity for both urine and blood analysis. Additionally, rapid procedures for both urine and blood analysis were established and compared. The two-step procedure at 1.0 mA/cm2 for 60 min followed by 1.56 mA/cm2 for 20 min increased the blotting efficiency compared with the commonly used constant current approach. Comparison of immunopurification revealed no significant difference in selectivity and sensitivity between the different methods. For other factors, such as operation complexity, time and cost, a StemCell® purification kit followed by single blotting and magnetic beads followed by double blotting are recommended for urine screening and confirmation, respectively. While magnetic beads and a MAIIA® kit followed by double blotting are recommended for both screening and confirmation of blood samples, respectively. To ensure high sensitivity and selectivity, double blotting is recommended for a rapid procedure for both urine and blood analysis.

Analysis of Protein Glycation Using Phenylboronate Acrylamide Gel Electrophoresis.

Carbohydrate modification of proteins adds complexity and diversity to the proteome. However, undesired carbohydrate modifications also occur in the form of glycation, which have been implicated in diseases such as diabetes, Alzheimer's disease, autoimmune diseases, and cancer. The analysis of glycated proteins is challenging due to their complexity and variability. Numerous analytical techniques have been developed that require expensive specialized equipment and complex data analysis. In this chapter, we describe two easy-to-use electrophoresis-based methods that will enable researchers to detect, identify, and analyze these posttranslational modifications. This new cost-effective methodology will aid the detection of unwanted glycation products in processed foods and may lead to new diagnostics and therapeutics for age-related chronic diseases.

A Multichannel Gel Electrophoresis and Continuous Fraction Collection Apparatus for High-Throughput Protein Separation and Characterization.

We developed a multichannel gel electrophoresis system that continuously collects fractions as protein bands migrate to the bottom of gel columns. The device uses several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "counter-free-flow" elution technique allows continuous and simultaneous fraction collection from multiple channels at low cost. Using the system with SDS-PAGE, 300 µg samples of protein can be separated and eluted into 48-96 fractions over a mass range of 10-150 kDa in 2.5 h. Each eluted protein can be recovered at 50% efficiency or higher in ~500 µL. The system can also be used for native gel electrophoresis, but protein aggregation limits the loading capacity to about 50 µg per channel and reduces resolution. This system has the potential to be coupled with mass spectrometry to achieve high-throughput protein identification.

Ultrarapid Sodium Dodecyl Sulfate Polyacrylamide Mini-Gel Electrophoresis.

We describe here an ultrafast method for electrophoresing proteins on SDS-PAGE. Previously we reported a method to complete SDS-PAGE and immunoblotting in an hour, including electrophoresing proteins at 70°C in 10 min. Here we show that we can electrophorese molecular weight standards and bovine serum albumin on a 4-20% gradient gel in well under 10 min using heated (44 °C) Laemmli running buffer and high voltage.

Serine Protease Zymography: Low-Cost, Rapid, and Highly Sensitive RAMA Casein Zymography.

To detect serine protease activity by zymography, casein and CBB stain have been used as a substrate and a detection procedure, respectively. Casein zymography has been using substrate concentration at 1 mg/mL and employing conventional CBB stain. Although ordinary casein zymography provides reproducible results, it has several disadvantages including time-consuming and relative low sensitivity. Improved casein zymography, RAMA casein zymography, is rapid and highly sensitive. RAMA casein zymography completes the detection process within 1 h after incubation and increases the sensitivity at least by tenfold. In addition to serine protease, the method also detects metalloprotease 7 (MMP7, Matrilysin) with high sensitivity.

Sensitive detection of proteins in polyacrylamide gel via isatoic anhydride derivatization: Introduction of a low-cost fluorescent prelabeling procedure.

Here, we introduce isatoic anhydride as a sensitive and commodious fluorescent prelabel for detection of proteins in one-dimensional polyacrylamide gels. High reactivity of isatoic anhydride with nucleophiles in mild alkaline environments makes it an appropriate tag for labeling of biomolecules. In this study, we show that preelectrophoresis labeling of proteins with isatoic anhydride for few minutes at room temperature allows detection of 2-4 ng of standard proteins, BSA and lysozyme, per band. Proteins were successfully labeled in the presence of a wide range of common biological reagents and in crude cell extract. The labeled proteins have the same electrophoretic migration in comparison to unlabeled proteins; however the application of saturation labeling method results in slight band broadening. Compatibility of the method with downstream processes was assessed by tryptic digestion of labeled proteins and study of peptide mixture using gel electrophoresis which revealed partial digestion of labeled proteins due to lysine modification. The present procedure is sensitive, rapid, and inexpensive and is a promising alternative for current protein staining procedures, where downstream processes are not desired.

Simple and cost effective apparatus for silver staining of polyacrylamide gels with sequential reagents addition and real time monitoring.

Highly reproducible results in molecular biology depend a lot on effective staining and destaining methods. Silver staining of polyacrylamide DNA and protein gel has been adopted widely in the molecular biology laboratories for detecting a very low nanogram range of sample. An efficient staining of a polyacrylamide gel requires a number of well controlled and highly sensitive steps that often becomes tiresome when done manually or when there are a number of gels to be stained simultaneously. Since, silver staining is a multistep procedure that requires proper fixation and exchange of substance, a reliable protocol is necessary and a simple apparatus may be an added advantage to carry out the steps with ease and safety. Here, we describe a simple and cost effective device made from off-the-shelf components for some established silver staining protocols. Staining is done on a tray while six graduated bottles with a liquid delivery stopcock each, is connected to the tray through silicon tubing. The used up solution is drained off completely from the staining tray through a liquid outlet stopcock using vacuum pressure. The system is fixed with a camera connected to a computer for effective control of the staining process in each step. The apparatus provides the researchers with efficient staining and real time monitoring of gels without the need for handling toxic chemicals.

Cost-effective and efficient apparatus for electroelution of micro- and macrogram quantities of proteins from polyacrylamide gels.

Protein recovery from gel electrophoresis plays a significant role in functional genomics and proteomics. To assist in this, a simple, cost-effective, and efficient apparatus for electroelution of proteins has been designed. The performance of the apparatus was demonstrated using the proteins bovine serum albumin (BSA), phosphorylase, ovalbumin, pepsin, and trypsinogen. In all the cases the yield of elution was found to be consistently greater than 85% and the proteins could be eluted without degradation in less than 15 min. The utility of this method can be extended to protein elution from denatured and native polyacrylamide gels, DNA purification from agarose gels, and oligomeric primers purification from polyacrylamide gels. In addition to this, the method offers an effortless purification and characterization of microbial extracellular proteins. The eluted proteins can be directly used in N-terminal amino acid sequencing, and in amino acid and proteomics analyses.

Development of buffers for fast semidry transfer of proteins.

Western blot is an extensively used method for protein detection in cell biology. To optimize this procedure, here we examined a panel of buffers for their ability to efficiently transfer proteins from SDS-polyacrylamide gels onto nitrocellulose membranes in a short 12-min period, designated here as fast semidry transfer. Our results show for the first time that HEPES- and HEPPS/EPPS-based buffers represent the most efficient buffers for fast semidry transfer.

Electroelution of nucleic acids from polyacrylamide gels: a custom-made, agarose-based electroeluter.

Polyacrylamide electrophoresis is routinely used for small-scale preparative and analytical separations. The incomparably high-resolution separations achieved by this technique, however, have not been widely exploited to the large-scale preparative isolation of biological molecules from contaminants, mainly because of difficulties in the recovery of the desired molecule from the gel matrix. Electroelution is an effective procedure applied for this purpose. However, commercially available, high-cost electroeluters are required for achieving high recovery yields. Here, we describe a custom-made electroeluter that combines low-cost, high-recovery yields, short times of electroelution, and convenience in the manipulation of sensitive samples.

An improved protocol for better detection of protein using 8-anilino-1-naphthalenesulfonate.

This study describes the optimum conditions at the staining time and the signal intensity for using 8-anilino-1-naphthalenesulfonate (ANS) as a fluorescent probe to detect proteins in SDS-PAGE. Using the optimized protocol, protein can be easily detected by short time fixing (20 min) and washing (2 × 5 min), followed by 10 min of staining. As low as 1-2 ng of the protein band can be detected, approximately thirty-fold higher than that of the original protocol. Furthermore, the compatibility of the staining method with MS was also explored by comparing the peptide mass fingerprinting results data of serial dilutions of BSA and ovalbumin stained by ANS with SYPRO Ruby.

Use of polyacrylamide gel moving boundary electrophoresis to enable low-power protein analysis in a compact microdevice.

In designing a protein electrophoresis platform composed of a single-inlet, single-outlet microchannel powered solely by voltage control (no pumps, values, injectors), we adapted the original protein electrophoresis format-moving boundary electrophoresis (MBE)-to a high-performance, compact microfluidic format. Key to the microfluidic adaptation is minimization of injection dispersion during sample injection. To reduce injection dispersion, we utilize a photopatterned free-solution-polyacrylamide gel (PAG) stacking interface at the head of the MBE microchannel. The nanoporous PAG molecular sieve physically induces a mobility shift that acts to enrich and sharpen protein fronts as proteins enter the microchannel. Various PAG configurations are characterized, with injection dispersion reduced by up to 85%. When employed for analysis of a model protein sample, microfluidic PAG MBE baseline-resolved species in 5 s and in a separation distance of less than 1 mm. PAG MBE thus demonstrates electrophoretic assays with minimal interfacing and sample handling, while maintaining separation performance. Owing to the short separation lengths needed in PAG MBE, we reduced the separation channel length to demonstrate an electrophoretic immunoassay powered with an off-the-shelf 9 V battery. The electrophoretic immunoassay consumed less than 3 µW of power and was completed in 30 s. To our knowledge, this is the lowest voltage and lowest power electrophoretic protein separation reported. Looking forward, we see the low-power PAG MBE as a basis for highly multiplexed protein separations (mobility shift screening assays) as well as for portable low-power diagnostic assays.

A rapid and simplified method for protein silver staining in polyacrylamide gels.

Silver staining is widely used to detect protein in polyacrylamide gels when high sensitivity is required. A simple and rapid protocol for silver staining of proteins following PAGE was developed in the present study. The number of steps was reduced compared to conventional protocol by combining fixing, rinsing, and soaking into a single impregnating step, thus achieving detection of proteins in 20 min. The present method is as sensitive as current protocols with the advantage of saving time and costs.

Simple, sensitive, and quick protocol to detect less than 1 ng of bacterial lipopolysaccharide.

Detection and quantitation of lipopolysaccharide (LPS) are important for quality assurance of pharmaceutical, biopharmaceutical products and for several research and industrial aspects. The widely available assays for LPS detection are the normal, or chromogenic, and the synthetic LAL (Limulus amebocyte lysate). Unfortunately, both assays are expensive and could not distinguish between different types of bacterial LPS, while LPS silver nitrate staining requires more than 20 hr and can only detect 5 ng LPS. The current modified protocol was able to detect less than 0.5 ng LPS in a polyacrylamide-urea gel. The procedure is rapid, inexpensive, and reproducible. It depends on introducing two modifications to improve the staining sensitivity in sample buffer and staining procedure. The results revealed that excluding the reducing agent sucrose to use instead glycerol, and replacing SDS with DOC, as well as incorporation of 4 M urea in stacking and separating gels, increased the sensitivity up to 150 pg. In summary, the gels were fixed, carbohydrates moieties were oxidized to create active aldehydes, and the gels were silver stained using non-ammoniacal silver nitrate.

A coordination complex system for generic, ultrafast, and sensitive multimode fluorescent staining of biomolecules.

Gel electrophoresis staining methodologies documented thus far are largely utilized in a biomolecule context-dependent manner. We report herein the development of a generic, ultrafast, and sensitive multimode fluorescent system for the efficient identification of DNA, RNA, and proteins. Interaction between a positively charged, planar ligand-based coordination complex with partner biomolecule leads to aggregation-induced fluorescence quenching and allows for the image contrast generation within one minute. Alternatively, successive reactions of the biomolecule-loaded gel with cation and ligand, in either order of sequence, provide an equally effective staining efficacy. Image contrast reversal is accomplished through a facile washing or photobleaching procedure. The versatility in the applicable target species and signal generation modes provides a hint at the design of novel staining structures and potentially enables the high-throughput readout of biomolecules.

High-resolution gel electrophoresis and sodium dodecyl sulphate-agarose gel electrophoresis on urine samples for qualitative analysis of proteinuria in dogs.

The aims of the current study were to assess whether sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE) and high-resolution electrophoresis (HRE) can identify dogs with a urinary protein-to-creatinine ratio (UPC ratio) >0.2 and whether HRE can provide preliminary information about the type of proteinuria, using SDS-AGE as a reference method. HRE and SDS-AGE were conducted on 87 urine samples classified according to the International Renal Interest Society as non-proteinuric (NP; UPC ratio: <0.20; 32/87), borderline proteinuric (BP; UPC ratio: 0.21-0.50; 15/87), or proteinuric (P; UPC ratio: >0.51; 40/87). SDS-AGE and HRE were positive in 14 out of 32 and 3 out of 32 NP samples and in 52 out of 55 and 40 out of 55 samples with a UPC ratio >0.20, respectively. The concordance between HRE or SDS and UPC ratio was comparable (κ = 0.59; κ = 0.55). However, specificity (90%) and positive likelihood ratio (7.76) were higher for HRE than for SDS-AGE (56% and 2.16) while sensitivity was lower (73% vs. 94%). The analysis of HRE results revealed that a percentage of albumin >41.4% and an albumin/α(1)-globulin ratio (alb/α(1) ratio) >1.46 can identify samples classified by SDS-AGE as affected by glomerular proteinuria while a percentage of α(1)-globulin >40.8% and an alb/α(1) ratio <0.84 can identify samples classified by SDS-AGE as affected by tubular proteinuria. In conclusion, both SDS-AGE and HRE could misclassify samples with a UPC ratio higher or lower than 0.20. Therefore, UPC ratio must always be determined before conducting these tests. The percentage of albumin and α(1)-globulin or the alb/α(1) ratio determined by HRE can provide preliminary information about the origin of proteinuria.

A cost-effective method for simultaneous homo-oligomeric size determination and monodispersity conditions for membrane proteins.

The use of blue native polyacrylamide gel electrophoresis (BN-PAGE) has been reported in the literature to retain both water-soluble and membrane protein complexes in their native hetero-oligomeric state and to determine the molecular weight of membrane proteins. However, membrane proteins show abnormal mobility when compared with water-soluble markers. Although one could use membrane proteins as markers or apply a conversion factor to the observed molecular weight to account for the bound Coomassie blue dye, when one just wants to assess homo-oligomeric size, these methods appear to be too time-consuming or might not be generally applicable. Here, during detergent screening studies to identify the best detergent for achieving a monodisperse sample, we observed that under certain conditions membrane proteins tend to form ladders of increasing oligomeric size. Although the ladders themselves contain no indication of which band represents the correct oligomeric size, they provide a scale that can be compared with a single band, representing the native homo-oligomeric size, obtained in other conditions of the screen. We show that this approach works for three membrane proteins: CorA (42 kDa), aquaporin Z (25 kDa), and small hydrophobic (SH) protein from respiratory syncytial virus (8 kDa). In addition, polydispersity results and identification of the most suitable detergent correlate optimally not only with size exclusion chromatography (SEC) but also with results from sedimentation velocity and equilibrium experiments. Because it involves minute quantities of sample and detergent, this method can be used in high-throughput approaches as a low-cost technique.

Gel electrophoresis and X-ray fluorescence: A powerful combination for the analysis of protein metal binding.

Understanding the complex biochemical mechanisms that underlie the regulation, toxicity, and protein binding of metal ions requires the ability to analyze the metal content of individual proteins in complex mixtures. In this issue of ACS Chemical Biology, a technique combining gel electrophoresis with synchrotron X-ray fluorescence imaging demonstrates a rapid and powerful solution for simultaneously examining multiple proteins and metal ions of interest. The resulting technique is broadly applicable, does not require specialized equipment for sample preparation, and is likely to be extensible in the future.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis for screening nonaggregating human antibody heavy chain variable domains.

Antibody heavy chain variable domains (V(H)s) form a significant class of biologics. With V(H) display libraries-the primary source of V(H) binders-unwanted aggregating V(H)s are coselected, sometimes overwhelmingly, alongside nonaggregating V(H)s. Thus, methods enabling efficient screening for nonaggregating V(H)s are highly valuable. Here, we found that on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, nonaggregating V(H)s migrate faster than expected, giving underestimated molecular weights (MWs), whereas aggregating ones migrate slower, giving overestimated MWs. Our finding can be applied to large-scale screening for nonaggregating V(H)s and possibly other proteins, in particular in display library settings, by SDS-PAGE.

High-resolution separation of tubulin monomers on polyacrylamide minigels.

High-resolution separation of alpha- and beta-tubulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on minigels can be performed rapidly using simple modifications of the standard Laemmli procedure. Separation of the subunits can be observed even in high-protein loads (up to 40microg of protein).