Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids

The objective of the present study was to develop a stain permitting automated quantification of myocellular lipid depositions in skeletal muscle sections together with immunolocalisation of other myocellular constituents by fluorescence microscopy. Lipid droplets were detected in skeletal muscle by oil red O (ORO). Conventional ORO was modified to diminish background staining, prevent crystallisation of ORO and to optimise lipid retention in cryosections. These modifications resulted in a punctate staining of lipid droplets, rather than the somewhat diffuse staining by conventional ORO. Small cavities in muscle sections (like the lumen of small blood vessels) lack ORO when using the protocol presented here. In addition a staining protocol is presented combining ORO with immunofluorescence. This combination permits multiple staining studies in the same section. Thus, lipid droplets can be studied together with immunolabelling of proteins involved in lipid handling and metabolism. This will extend our knowledge on the subcellular localisation of lipid handling proteins (i.e. enzymes and fatty acid transporting proteins) in relation to the localisation of lipid depositions. In conclusion, the protocol presented here permits examination of ORO-stained lipid droplets in skeletal muscle sections together with multiple staining of other immunodetectable proteins present in skeletal muscle by quantitative fluorescence microscopy.     

A fluorescence-based Coomassie Blue protocol for two-dimensional gel-based proteomics

A sensitive and convenient “visible SYPRO” staining protocol was developed for visualizing proteins after SDS-PAGE. Gels were sensitized with SYPRO Ruby and then stained with the Coomassie Brilliant Blue G-250 protocol (Blue Silver). This combined protocol had similar or better linearity than staining with only SYPRO Ruby or Blue Silver, respectively. In addition, this method was more sensitive than that of Blue Silver, simpler than that of SYPRO Ruby, and compatible with subsequent mass spectrometry analysis.     

A modified Coomassie Brilliant Blue staining method at nanogram sensitivity compatible with proteomic analysis

A more sensitive and convenient Coomassie Brilliant Blue (CBB) staining method for visualizing proteins was developed. Compared with the modifications include the supplement of 10% (v/v) methanol into the fixing solution, an increase of an additional sensitization step and CBB raised from 0.1 to 0.125%. The improved method can detect proteins at nanogram level. The improved method is more sensitive than Blue Silver and more convenient than the Silver protocol. Mass spectrometry results confirmed that it is suitable for subsequent proteomic research.     

A simple and cost-effective solid-phase protein nano-assay using polyacrylamide-coated glass plates

A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from conventional “on-filter” assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of “on-membrane” staining with the sensitivity and ease of documentation of “in-gel” staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2 ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required.     

Evaluation of protocols used in 2-d electrophoresis for proteome analysis of young rice caryopsis

In order to obtain a high-resolution electrophorogram of rice young panicle proteome, we evaluated various protocols commonly used in two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) of proteins, including gel staining protocol, pH range of immobilized pH gradient (IPG) strips and sample loading quantity. Results showed that a silver staining protocol using sensitized solution containing glacial acetic acid, sodium acetate and sodium thiosulfate (reported by Heukeshoven and Dernick in 1988) and a Coomassie Brilliant Blue staining method using solution containing G-250, ammonium sulfate and phosphoric acid (reported by Pink et al in 2010) demonstrated the superior staining effect. In addition, we also showed that higher resolution was achieved when IPG gel strip with pH range of 5-8 was used, compared to that with pH range of 4-7. Finally, the optimal loading quantity was determined as 130 μg using the 17 cm-long nonlinear IPG strip with pH 5-8 in combination with the silver nitrate staining protocol. The evaluated results would be helpful in proteome analysis of young rice caryopsis.     

Eosin Y staining of proteins in polyacrylamide gels.

A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins.     

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa^1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol ³ . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography^4,5. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples ⁶. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.     

Improved immunocytochemical staining of carcinogen-DNA adducts by a capillary slot block system.

We developed an immunocytochemical protocol in which incubation occurs in a capillary slot instead of the conventional horizontal drop. Slots of constant width were formed by placing slides on top of each other with parafilm spacer layers in between. Cryostat or semi-thin plastic-embedded sections were cut from organs of carcinogen-treated experimental animals. Carcinogen-DNA adducts were visualized in the affected nuclei by a double peroxidase-antiperoxidase method using rabbit antisera specific for certain DNA adducts formed. The staining in capillary slot blocks offered better staining reproducibility than the conventional method. This is particularly important when the staining intensity must be quantified. In addition, handling of the blocks was substantially less laborious than the individual treatment of slides, making this protocol especially suitable for larger series of slides. Other applications for the capillary slot block protocol should be enzyme histochemistry and in situ hybridization.     

About the mechanism of interference of silver staining with peptide mass spectrometry

The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde-free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.     

BrdU related direct revelation of typical dynamic R- and G-banding with the use of monoclonal anti-BrdU antibody.

Pulse 5-bromodeoxyuridine (5-BrdU) incorporation during the last S-phase is known to produce R- or G-banded chromosomes after photolysis-plus-Giemsa (FPG) staining. The authors applied an immunological staining with monoclonal anti-BrdU antibody instead of the FPG protocol. The results offered banded chromosomes with an immunological typical R-banding (RBI) on the GBG cultivated cells (early pulse incorporation), and an immunological G-banding (GBI) on the RBG cultivated ones (late pulse incorporation). After a further FPG protocol following an immunological treatment, an inverted banding pattern became evident whereas a faint immunological staining remained. Thus the method superimposed a GBG-banding on the RBI-staining or a RBG on the GBI one. This allows a rapid and easy R and G double chromosomal identification on the same metaphase cell, using first the immunological banding then the classical FPG staining. The method allows a reproducible dynamic G-banding with an easy monitored late 5-BrdU pulse incorporation specially attractive in spontaneous dividing cells from bone marrow. This dynamic G-banding protocol should be extended to chorionic villi and malignant cells. Our data are in agreement with a connection between dynamic banding and chromosomal portions containing or not BrdU. The lack of an immunological staining after the FPG protocol has been noticed and assume the photolysis degradation-elution of the DNA in BrdU-substituted areas.     

An improved method for rapid preparation of oligodendrocyte-specific rabbit polyclonal antibody

Antibodies are important tools in the study of protein function and diagnostic tests. However, traditional antiserum preparation requires a time-consuming immunization protocol and subsequent purification of polyclonal antibodies. In this study, a rapid and efficient method for polyclonal antibody preparation has been developed. Juxtanodin (JN) and silent information regulator-2 (Sirt2), both of which are oligodendrocyte-specific proteins, were used for antibody preparation. The N-terminal 170 amino acids of JN (JN170) and amino acids 231–351 of Sirt2 (Sirt2-121) were expressed as GST-tagged proteins from a pET-41a(+) vector in E. coli strain BL21 (DE3) cells. The fusion proteins were purified and used to immunize rabbits following both a traditional protocol, in which antigen was presented biweekly, and a modified rapid protocol, in which the immunization on day 1 was boosted on days 5 and 28. ELISA, Western blot analysis and immunofluorescent staining showed that antibodies produced via the rapid protocol could recognize these two oligodendrocytespecific proteins in vitro and in the rat central nervous system (CNS), respectively, similar to those produced with the traditional protocol. Thus, our study provides a novel rapid method to prepare high specificity antibodies via a modified immunization protocol and subsequent antibody purification.     

In silico chaperonin-like cycle helps folding of proteins for structure prediction

Currently, one of the most serious problems in protein-folding simulations for de novo structure prediction is conformational sampling of medium-to-large proteins. In vivo, folding of these proteins is mediated by molecular chaperones. Inspired by the functions of chaperonins, we designed a simple chaperonin-like simulation protocol within the framework of the standard fragment assembly method: in our protocol, the strength of the hydrophobic interaction is periodically modulated to help the protein escape from misfolded structures. We tested this protocol for 38 proteins and found that, using a certain defined criterion of success, our method could successfully predict the native structures of 14 targets, whereas only those of 10 targets were successfully predicted using the standard protocol. In particular, for non-α-helical proteins, our method yielded significantly better predictions than the standard approach. This chaperonin-inspired protocol that enhanced de novo structure prediction using folding simulations may, in turn, provide new insights into the working principles underlying the chaperonin system.     

Silver staining DNA in polyacrylamide gels

This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (approximately 1 h) and is implemented using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts.     

A sensitive urea-silver stain method for detecting trace quantities of separated proteins in polyacrylamide gels

An ultrasensitive method using a urea-silver staining procedure to detect trace quantities of proteins in polyacrylamide gels (PAGE) is described. This technique is sensitive enough to detect picogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels. The major advantages of our method are that it provides a clear background and it is more sensitive than other techniques allowing it to substitute for radioisotopic techniques in some cases.     

Tricine-SDS-PAGE

Tricine-SDS-PAGE is commonly used to separate proteins in the mass range 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine-SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1-2 d.     

An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells

Intracellular staining is a widely used flow cytometry (FCM)-based technique to detect the expression of cytoslio nucleic antigens. However, intracellular staining of cells expressing cytosolic fluorescent protein (FP) markers was proven to be problematic as significant loss of the FP-signal was routinely observed. Using splenocytes harvested from mice constitutively expressing the enhanced yellow fluorescent proteins (YFP) as a model, we modified the widely used intracellular staining protocol and successfully achieved simultaneous detection of both the nuclear proteins and YFP in T-regulatory cells. The improved protocol can be used to perform antibody-based intracellular characterization of FP-labeled target cells, while maintaining their fluorescent reporter signals for easy tracing and identification.     

A method for in-gel fluorescent visualization of proteins after native and sodium dodecyl sulfate polyacrylamide gel electrophoresis

We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation–emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide.     

Daily changes in the phosphoproteome of the dinoflagellate Lingulodinium

The dinoflagellate Lingulodinium has a large number of daily rhythms, many of which have no biochemical correlates. We examined the possibility that changes in protein phosphorylation may mediate some of the rhythmic changes by comparing proteins prepared from midday (LD6) and midnight (LD18) cultures. We used two different methods, one a 2D gel protocol in which phosphoproteins were identified after staining with ProQ Diamond, and the other an LC-MS/MS identification of tryptic phosphopeptides that had been purified by TiO(2) chromatography. Two differentially phosphorylated proteins, a light harvesting complex protein and Rad24, were identified using the 2D gel protocol. Six differentially phosphorylated proteins, a polyketide synthase, an uncharacterized transporter, a LIM (actin binding) domain and three RNA binding domain proteins, were identified using the phosphopeptide enrichment protocol. We conclude that changes in protein phosphorylation may underlie some of the rhythmic behavior of Lingulodinium.     

Cytological staining of protozoa: a case study on the impregnation of hypotrichs (Ciliophora: spirotrichea) using laboratory-synthesized protargol

Protargol (silver proteinate) impregnation is a common method used to identify and characterize ciliated protozoa. Unfortunately, chemical companies have stopped producing the ‘strong’ protargol powder used in this method. Based on an in-house protocol for its synthesis published in 2013, more than 10 batches of protargol powder were produced and subsequently applied in taxonomic studies. During these studies, the protocol for protargol powder synthesis was slightly modified and employed a peptone not originally listed in the 2013 protocol. This modification improved the results of the impregnation protocol. Protargol preparations of hypotrichs were optimized by adjusting the pH during staining rather than during the synthesis. The pH was adjusted to 7.5–7.6, and an acetone developer was used. While the conditions used in this study are not completely comparable to those using the commercially produced protargol, access to this information could help researchers investigate the diversity of ciliates, particularly hypotrichs.