Rapid and easy protein staining for SDS-PAGE using an intramolecular charge transfer-based fluorescent reagent

High-performance staining for 1-D and 2-D SDS-PAGE was carried out using a novel protein-binding fluorophore (Dye 1), which noncovalently interacts with proteins and provides a fluorescence emission response to proteins by intramolecular charge transfer. In order to achieve the high-throughput analysis of proteins for SDS-PAGE, the general protocols for in-gel protein staining (SDS-PAGE, fixation, staining, washing, and detection) were simplified to produce an easy and rapid protocol (SDS-PAGE together with staining, washing, and detection). This method was performed by preparation of an electrophoresis buffer containing Dye 1 under optimum conditions, and by the binding of Dye 1 to proteins in the gel during the SDS-PAGE. As a result, this study required only 15 min for protein staining as a minimum time. On the other hand, it takes several hours for the general protein staining method, such as SYPRO Ruby staining (18 h) and CBB staining (105 min). Moreover, the protein-to-protein variation was low, and the detection limit was 7.0 ng/band of BSA (S/N = 3.0) in this method, which was as sensitive as the short-protocol silver staining methods. On the basis of these results, this rapid and easy protocol for SDS-PAGE using Dye 1 may be widely applicable and convenient for users in the various scientific and medical fields.     

Improvement of gel‐separated protein identification by DMF‐assisted digestion and peptide recovery after electroblotting

In‐gel digestion of gel‐separated proteins is a major route to assist in proteomics‐based biological discovery, which, however, is often embarrassed by its inherent limitations such as the low digestion efficiency and the low recovery of proteolytic peptides. For overcoming these limitations, many efforts have been directed at developing alternative methods to avoid the in‐digestion. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel‐separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (≥99.8%). Before tryptic digestion, NH₄HCO₃ buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane‐bound substances, the proteins were virtually in‐solution digested in DMF‐containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane‐enriched sample showed that the method was superior to the reported on‐membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.     

Discontinuous native protein gel electrophoresis

Analysis of the oligomeric state of a native protein usually requires analytical ultracentrifugation or repeated gel filtration to calculate the protein's size. We have developed a discontinuous native protein gel electrophoresis system that allows the separation of even basic proteins according to their size, oligomeric state, and shape. This gel system combines the addition of negative charges to the proteins by Serva Blue G with a discontinuous buffer system and gradient gels. As in SDS-PAGE, chloride constitutes the high mobility anion in the gel and anode buffer. However, for sample focusing this system employs histidine instead of glycine as the slow dipolar ion following from the cathode buffer to improve migration of basic proteins. In addition, proteins run into gel pores corresponding to their size and shape in the gradient gel. Using this gel system, we show that the polypyrimidine tract-binding protein (PTB) is a monomer.     

A comparison of Tris-glycine and Tris-tricine buffers for the electrophoretic separation of major serum proteins

This paper compares different buffer systems for the electrophoretic separation of the five most abundant serum proteins on native-PAGE gel and cellulose membranes. A modified Tris-tricine system was shown to be superior for the separation of these serum proteins in a 7% m/v native-PAGE gel as compared with the traditionally used Tris-glycine and Tris-tricine methods. This modified Tris-tricine buffer system was also employed for the separation of serum proteins using a cellulose acetate membrane and very effective separation was observed as compared with the traditionally used Tris-barbital and Tris-glycine buffer systems.     

In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.     

Cell surface proteins of Candida albicans: preparation of extracts and improved detection of proteins

We have reexamined the detection of the components in a beta-mercaptoethanol and ammonium carbonate buffer extract of surface proteins of Candida albicans and the effects of postextraction manipulation of the extract on recovery of extract components. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), preferential staining of some moieties was observed when bands detected by a commercial silver staining method or a Coomassie Brilliant Blue (CBB) staining method were compared. Additional protein bands that were either not detected or poorly detected by a single method alone were readily observed by a combined silver-CBB staining method. This method also detected alterations in the profile of extracted proteins from organisms grown in the presence of galactose or hemoglobin rather than glucose. Two-dimensional electrophoresis (2-DE) gel analysis by double stain showed better detection of several acidic and basic protein spots. Less than 10% of the extract as determined by a dye-binding assay was lost following either or both lyophilization and dialysis. These manipulations of the extract did not change the protein profile following SDS-PAGE as determined by the combined staining or Western blot analysis of a 70 kDa protein. These observations suggest that soluble cell wall proteins are not unusually sensitive to procedures routinely used in protein purification. In addition, these studies suggest that a modified staining method that combines both silver stain and CBB stain provides improved detection of cell wall proteins compared to either method alone.     

Separation of membrane proteins by two-dimensional electrophoresis using cationic rehydrated strips

Due to their poor solubility during IEF membrane proteins cannot be separated and analyzed satisfactorily with classical 2-DE. A more efficient method for such hydrophobic proteins is the benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE, but the corresponding protocol is intricate and time-consuming. We now developed an easy-to-handle electrophoresis method in connection with a novel device which enables reproducible separation of ionic solubilized membrane proteins using individually rehydrated plastic sheet gel strips. These strips are suitable for the first dimension in a 2-D 16-BAC/SDS system and can be handled easily; this is demonstrated by the separation of membrane proteins of human embryonic kidney (HEK293) cells.     

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.     

Dialysis-assisted two-dimensional gel electrophoresis

2-DE is an important tool in proteomics research. However, intrinsic gel-to-gel variability of 2-DE often masks the biological differences between the samples and compromises quantitative comparison of protein expression levels. Here, we describe a modification of 2-DE that results in improved matching and quantification of proteins. This was accomplished by performing IEF of two samples in two IPG strips separated by a dialysis membrane. After IEF running, the strips were separated and the SDS-PAGE dimension was accomplished on two individual gels. After gel staining with CBB, ImageMaster 2D Platinum software (Amersham) was used for spot detection and quantification. Analysis of protein extracts from C2C12 myoblasts by this method resulted in 99% spot-matching efficiency and CV in stain intensity (% volume) was less than 0.5 for 98% of spots. We conclude that this technique, called dialysis-assisted gel electrophoresis, gives superior spot matching and quantitative reproducibility compared to IEF conducted on separate strips.     

多维离子交换色谱分离和串联质谱鉴定在小鼠肝脏膜蛋白质组分析中的应用

膜蛋白质在变性剂作用下能够较充分地溶解。根据这一特点,我们试图在变性剂溶液中采用串联离子交换色谱法分离小鼠肝脏膜蛋白质。将小鼠肝脏膜蛋白质溶解于含有4 mol/L尿素,20 mmol/L三羟甲基氨基甲烷(Tris)-盐酸缓冲液(pH 9.0)中,用Q-Sepharose FF和Sephacryl S-200HR树脂组成的色谱柱结合大部分溶解的膜蛋白质,然后采用氯化钠线性梯度洗脱蛋白质,分步收集后采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进一步分离洗脱组分的蛋白质。利用胶内胰蛋白酶消化技术将SDS-PAGE胶内分离的蛋白质降解为相应的肽段,然后以反相高效液相色谱分离和离子阱质谱仪鉴定肽段。根据文献报道和蛋白质的功能分类,在所鉴定的392个蛋白质中有306个可能为膜蛋白质或膜结合蛋白质。蛋白质的疏水性计算表明,GRAVY(grand average of hydropathicity)得分大于或等于0.00的蛋白质有83个。综上所述,我们有理由认为本实验方法基本符合小鼠肝脏膜蛋白质组学研究的要求。     

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.     

High yield electroblotting onto polyvinylidene difluoride membranes from polyacrylamide gels.

Optimal conditions of electroblotting that led to high protein recovery on polyvinylidene difluoride (PVDF) membranes were determined for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS concentrations in the gel and transfer buffer were found to be the most important factors affecting the amount of protein recovered on the PVDF membrane. The largest loss occurred during the first 10-30 min of transfer due to the relatively high initial SDS concentration in the gel. During this initial stage of transfer, most of the protein passed through the primary membrane and was partially retained on secondary and tertiary membranes. The value of presoaking gels prior to transfer to reduce the amount of SDS was evaluated by quantitating free SDS densitometrically and by correlating the reduced SDS concentration with increased electroblotting efficiency from presoaked gels. Transfer time was evaluated and no "overtransfer" was found even after very long transfer times. These results clearly indicate that proteins electroblotted onto PVDF membranes were tightly bound and could not be released by extending the transfer time. The effects of methanol and SDS concentrations on protein adsorption from solution to PVDF were also determined quantitatively. The results of this study strongly suggest that proteins fully saturated with SDS cannot bind efficiently to PVDF membranes. Since SDS is necessary for high protein mobility, the challenge in efficient electroblotting is to maintain an optimal SDS concentration which is high enough to permit effective removal from the gel and low enough to permit effective binding to the PVDF membrane. For 1.5 mm thick gels containing 0.2% SDS, presoaking the gel for 15-20 min in transfer buffer with 10% methanol prior to electroblotting provided the best recovery on the primary membrane.     

Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing

We have elaborated a protocol for the fractionation of both hydrophilic and hydrophobic proteins using as a model the matrix and membrane compartments of highly purified rat liver peroxisomes because of their distinct proteomes and characteristic composition with a high quota of basic proteins. To keep highly hydrophobic proteins in solution, an urea/thiourea/detergent mixture, as used in traditional gel-based isoelectric focusing (IEF), was added to the electrophoresis buffer. Electrophoresis was conducted in the ProTeam free-flow electrophoresis (FFE) apparatus of TECAN separating proteins into 96 fractions on a pH 3-12 gradient. Consecutive sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that both matrix and the integral membrane proteins of peroxisomes could be successfully fractionated and then identified by mass spectrometry. This is documented by the detection of PMP22, which is the most hydrophobic and basic protein of the peroxisomal membrane with a pI > 10. The identification of 96 prominent spots corresponding to polypeptides with different physical and chemical properties, e.g., the most abundant integral membrane polypeptides of peroxisomes and specific ones of the mitochondrial and microsomal membrane, reflects the fractionation potential of free-flow (FF)-IEF, accentuating its value in proteomic research as an alternative perhaps superior to gel-based IEF.     

Requirements for the application of protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis and randomly amplified polymorphic DNA analyses to product speciation

Raw, cooked, fried, smoked and gravad (brine-cured) products were analyzed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins and by randomly amplified polymorphic DNA (RAPD) in order to identify the species used in their manufacture. The discriminatory power of SDS-PAGE was dependent primarily on the composition and secondarily on the size of the gels: the Laemmli buffer system with 15% acrylamide and 0.087% piperazine diacrylamide separating gels resolved more discriminant protein bands than any of the commercial gels tested. Some of the processing conditions induced alterations in the protein patterns that made identification dubious. Differentiation even between closely related species was easier by RAPD than by SDS-PAGE. Neither the processing conditions nor the tissue from which the DNA was extracted had a significant effect on the RAPD profiles. For identifications based on SDS-PAGE, one should use an optimized gel composition and separate the sample under analysis in the same gel as the references. For RAPD-based identifications, the unknown sample should be amplified together with reference samples and separated in the same gel.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2-10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 +/- 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma.     

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) still remains the most reliable and comprehensive analytical method for the evaluation of protein extracts. However, conventional SDS-PAGE is time-consuming and, thus, unpractical if several tens or hundreds of samples must be examined. We show that SDS-PAGE protein analysis can be automated using slab gel DNA sequencers and compare the instrument's performance with conventional SDS-PAGE in terms of resolution, sensitivity and sample capacity. Labeled protein bands are detected online by laser-induced fluorescence (LIF) and the acquired signals are electronically stored for further processing, avoiding gel staining and scanning. Appropriate software allows immediate display of recorded data and convenient evaluation. The method provides a higher sensitivity and dynamic range than conventional Coomassie-stained gels and the resolution of proteins with different masses is independent of the polyacrylamide concentration. Internal markers can also be used for direct quantification and assignment of the molecular masses. Additionally, we present a novel electrophoresis instrument for the simultaneous separation and online LIF detection of all samples of a microtiterplate in parallel lanes in a 3-D geometry gel cylinder. The specific gel thermostatting concept prevents irregular sample migration (smiling) and improves the reproducibility and comparability of individual separation patterns. In combination with the expected large capacity of 384 or 1,536 samples, this makes the instrument a valuable tool for high-throughput comparative screening applications.     

Protein extraction for 2-DE from the lamina of Ecklonia kurome (laminariales): recalcitrant tissue containing high levels of viscous polysaccharides

Extraction of proteins from the tissues of laminarialean algae, i.e. kelp, is difficult due to high levels of nonprotein interfering compounds, mainly viscous polysaccharides. To establish proteomic analysis of kelp species, an ethanol/phenol extraction method was developed and compared to other popular methods. Proteins were extracted with phenol from crude protein powder, obtained by homogenizing the kelp tissues in ice-cold ethanol. The ethanol/phenol method produced high-quality proteins of the highest purity from the lamina of Ecklonia kurome, one of the Japanese dominant laminarialean algae. This method gave well-resolved 1-D SDS-PAGE or 2-DE images with low background and the highest number of bands or spots. In particular, proteins with neutral to basic pI's were efficiently extracted. Furthermore, 27 spots on the 2-DE gel were extensively identified by MALDI-TOF/TOF analysis. To the best of our knowledge, this is the first report of a protocol for protein extraction from kelp tissues that gives satisfactory 2-D protein profiles. It is expected that the protocol can be applied to other algae tissues or other recalcitrant plant tissues containing high levels of nonprotein interfering compounds.     

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.     

Electroelution without gel sectioning of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis: fluorescent detection, recovery, isoelectric focusing and matrix assisted laser desorption/ionization-time of flight of the electroeluate

A method of direct electroelution of intact proteins, without gel sectioning and orthogonal to the orientation of electrophoretic migration, was developed in application to Novex gels, using a simple home-made experimental setup. Six model proteins covering the molecular mass range of 14-120 kDa were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with an aqueous solution of the fluorescent dye, SYPRO-red, and electroeluted from the intact gel. The sensitivity of visual detection was 0.1-0.2 microg upon illumination by a green laser and 0.5-1 microg of protein on side-ways UV-illumination. Duration (for each protein) and field strength were optimized to render protein electroelution from the gel near-quantitative (above 80%) and relatively fast (1-12 min at 1 kV). At a given field strength, the optimal duration was found to be inversely proportional to the mobility of proteins in SDS-PAGE. Sequential ultrafiltration was evaluated as a simple approach to concentrate electroeluted proteins and deplete SDS to a level compatible with mass spectrometry of proteins: protein yields in the electroeluate were 25-33% (depending on the protein used) after three steps of ultrafiltration with water. The analysis of the electroeluate by isoelectric focusing in an immobilized pH gradient, to reveal protein heterogeneity under a single SDS-PAGE band (prior, e.g., to mass spectrometric analysis), was demonstrated.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2– 10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 ± 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma. Received: 28 June 1999 / Revised: 14 September 1999 / Accepted: 16 September 1999