We thank Dr. Nicola Wieth&ouml;lter for preparing and staining of 1-D gels.
This work was funded by a grant from the Deutsche Forschungsgemeinschaft (GRK 1089/project 5 to ND and SM) and supported by a research fellowship from the J&uuml;rgen Manchot Stiftung to ND.

Part 1: Two-dimensional (2-D) gel electrophoresis using cup-loading
<ol>
<li>IPG-strip rehydration and isoelectric focusing (IEF) 
<ul>
<li>Rehydrate Immobiline DryStrip gels, pH 6-11 (7 cm) in 125 &mu;l rehydration solution [7 M urea, 2 M thiourea, 4% CHAPS, 50 mM hydroxyethyldisulfide and 2% IPG Buffer pH 6-11] using Immobiline DryStrip Reswelling Tray for at least 10 hours.</li>
<li>Dissolve precipitated protein sample in IEF sample buffer [7 M urea, 2 M thiourea, 2% CHAPS, 2% ASB-14, 50 mM hydroxyethyldisulfide and 2% IPG Buffer pH 6-11] corresponding to 60-100 &mu;g protein per 100 &mu;l.</li>
<li>After solubilization (at least 30 minutes at room temperature), apply protein sample via anodic cup-loading using sample cups. The volume of the cup is 100 &mu;l (Manifold cups allow up to 150 &mu;l). Note: you can load larger sample amounts if you insert a low-voltage step at the beginning of the focusing protocol and refill the cups while there is still a liquid film in the cup!</li>
<li>Perform isoelectric focusing for 11.1 kVh in gradient mode in the Multiphor II Electrophoresis Unit.</li>
</ul>
</li>
<li>Equilibration and SDS-PAGE 
<ul>
<li>After IEF, the IPG strips were subjected to reduction and alkylation, each time 15 minutes on a shaker, using 1% dithiothreitol and 2.5% iodoacetamide respectively in equilibration solution [50 mM Tris-HCl/pH 8.8, 6 M urea, 30% glycerol and 2% SDS]. </li>
<li>Rinse the equilibrated strips with H2O and blot them onto whatman paper to remove excess equilibration buffer. Afterwards dip the strips in SDS-running buffer (1X). </li>
<li>Second dimension is performed by SDS-PAGE on a vertical electrophoresis systems with a total acrylamide concentration of 12 %. The equilibrated IPG strips were placed on the top of the separating gels and fixed with hot agarose solution [0.5% agarose in running buffer containing bromophenol blue].</li>
<li>Proteins were separated for 2.5 hrs, starting at 80 V for 15 minutes followed by 120 V until the dye front reaches the bottom of the gel cassette.</li>
</ul>
</li>
</ol>
Part 2: Colloidal Coomassie staining with CBB G-250
1. Staining solutions Note: for the preparation of the staining solutions, use chemicals with high quality such as analytical grade (p.a.) and water of high purity as you get from Millipore systems (Milli-Q water).
<table border="0">
<tbody>
<tr>
<td colspan="2">Coomassie solution:</td>
<td>ad 2000 ml H2O</td>
</tr>
<tr>
<td>0.02 % (w/v)</td>
<td>CBB G-250</td>
<td>0,4 g</td>
</tr>
<tr>
<td>5 % (w/v)</td>
<td>aluminum sulfate-(14-18)-hydrate</td>
<td>100 g</td>
</tr>
<tr>
<td>10 % (v/v)</td>
<td>ethanol (96%)</td>
<td>200 ml</td>
</tr>
<tr>
<td>2 % (v/v)</td>
<td>orthophosphoric acid (85 %)</td>
<td>47 ml</td>
</tr>
</tbody>
</table>
Note: for the preparation of the staining solution, the sequential addition of the components in the following order has to be maintained:
<ol>
<li>first dissolve aluminum sulfate in Milli-Q water</li>
<li>thereafter add ethanol, homogenize, and mix CBB G-250 to the solution</li>
<li>as recently as the solution is completely dissolved, add phosphoric acid (the incorporation of the acid to the alcoholic media lets the Coomassie molecules aggregate into their colloidal state)</li>
<li>finally fill up with Milli-Q water</li>
</ol>
Note: the final staining solution has a dark green-bluish appearance and is full of particles swimming around:
<ul>
<li>Don't filtrate this solution!</li>
<li>If you changed the order of preparation, you would get a more violet-bluish solution that is less sensitive.</li>
</ul>
<table border="0">
<tbody>
<tr>
<td colspan="2">Destaining solution:</td>
<td>ad 2000 ml H2O</td>
</tr>
<tr>
<td>10 % (v/v)</td>
<td>ethanol (96 %)</td>
<td>200 ml</td>
</tr>
<tr>
<td>2 % (v/v)</td>
<td>orthophosphoric acid (85 %)</td>
<td>47 ml</td>
</tr>
</tbody>
</table>
2. Staining procedure
<ul>
<li>After second dimension separation, carefully remove the gels from the glass plates and transfer them into a staining dish filled with Milli-Q water.</li>
<li>Wash the gels three times with Milli-Q water for 10 minutes on a horizontal shaker (insufficient washing causes poor sensitivity because the remaining SDS on the gels disturbs the bounding of the dye to the protein).</li>
<li>Shake the Coomassie solution before use to disperse the colloidal particles evenly.</li>
<li>Incubate the gels well covered with the Coomassie solution by agitation on a shaker for 2-12 hours. This staining generates marginal background so you can observe the staining progress in between. Note: after 10 minutes you can see first protein spots appearing, within 2 hours of incubation about 80% staining to its maximum level is completed. For nearly 100% staining, overnight incubation is recommended. In some cases, when the amount of protein to be stained is huge and the solution turns into a bright blue, a refresh of the staining solution is necessary.</li>
<li>After the staining procedure remove the Coomassie solution and rinse the gels twice with Milli-Q water. Note: you can reuse the staining solution as long as particles are still remaining, otherwise discard it (keep in mind that your laboratory may have special regulations for waste disposal).</li>
<li>Remove sticking dye particles from the staining dish with a lint-free paper towel and destain for 10- 60 minutes. Note: You can also wash the gels just with Milli-Q water but this we recommend only for preparative gels because there will last an irregular, weak Coomassie film on the gels that will arise during the scanning procedure.</li>
<li>Finally rinse the gels twice with Milli-Q water: the gels will reach their original thickness (the alcohol-acid media makes the gels shrinking) and neutralization in water additionally enhances Coomassie's color intensity.</li>
</ul>
Part 3: Representative results
If you follow the protocol described above you will get on your 2-D gel distinctive resolved and well stained dark blue protein spots. Even compared to a 2-D gel stained with silver according to Shevchenko et al.5 ,a protocol claiming good sensitivity and compatibility with mass spectrometry, we achieve equal staining results.
<img src="/files/ftp_upload/1431/fig1.jpg" alt="Figure 1" /> Figure 1: Final outcome of the experimentation described above. Kang's Coomassie protocol (A) performs strongly like the silver staining according to Shevchenko et al. (B) in detecting proteins after 2-D gel electrophoresis.
Part 4: Tips and tricks
<ul>
<li>Perform some test stainings before processing your own samples. For unexplainable reasons we reported sometimes that a protein of interest could not be detected successfully even when milligrams were applied. In this case we recommend to prepare some dilution series to test sensitivity.</li>
<li>If you have low in-gel detection of protein(s) in general, unsuccessful transfer from the stacking to the separating gel during SDS-PAGE will possibly be the reason. You can check for such sticky proteins by staining the complete gel including the stacking area.</li>
<li>For a fast verification of your isoelectric focusing results, you can directly stain the IPG-strips without any further second dimension separation.</li>
<li>Occasionally turn the gels during the staining procedure so they are stained evenly from both sides (if you keep the gels unmoved, the dye just binds to one side of the gel). </li>
<li>We experienced that storage of the staining solution in a dark bottle increases its lifetime (in transparent bottles up to 4 month). </li>
<li>After staining and documenting you can keep the gels in the fridge for several years (if you add acidic solution you avoid contaminations by fungi). </li>
<li>Destaining of gel plugs for a tryptic digest can be accelerated by warming in a block heater.</li>
</ul>

<ol>
	<li>Fazekas de St Groth, S., Webster, S. R. G., Datyner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+new+staining+procedures+for+quantitative+estimation+of+proteins+on+electrophoretic+strips.">Two new staining procedures for quantitative estimation of proteins on electrophoretic strips.</a> Biochim. Biophys. Acta. 71, 377-391 (1963).</li><li>Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+staining+of+proteins+in+polyacrylamide+gels+including+isoelectric+focusing+gels+with+clear+background+at+nanogram+sensitivity+using+Coomassie+Brilliant+Blue+G-250+and+R-250.">Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.</a> Electrophoresis. 9, 255-262 (1988).</li><li>Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B., Orecchia, P., Zardi, L., Rigetti, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+Silver:+A+very+sensitive+colloidal+Coomassie+G-250+staining+for+proteome+analysis.">Blue Silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis.</a> Electrophoresis. 25, 1327-1333 (2004).</li><li>Kang, D., Gho, S. G., Suh, M., Kang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+and+Fast+Protein+Detection+with+Coomassie+Brilliant+Blue+in+Sodium+Dodecyl+Sulfate-Polyacrylamide+Gel+Electrophoresis.">Highly Sensitive and Fast Protein Detection with Coomassie Brilliant Blue in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.</a> Bull. Korean Chem. Soc. 11, 1511-1512 (2002).</li><li>Shevchenko, A., Wilm, M., Vorm, O., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+sequencing+of+proteins+silver-stained+polyacrylamide+gels.">Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.</a> Anal. Chem. 68, 850-858 (1996).</li></ol>

I have nothing to disclose.

Innovative or just another Coomassie protocol?
At the moment there exist multiple protocols for staining procedures with Coomassie Brilliant Blue. Most of them result from minor or major modifications of one of the most commonly used protocols by Neuhoff and colleagues2. Also Kang's protocol based on Neuhoff's formula. But is it really an alternative Coomassie staining method for proteomic research? We will picture two main issues, detection limit and usability, to evidence that it...

<table><tbody><tr><td>Immobiline DryStrip gels</td><td>GE Healthcare</td><td>17-6001-94</td><td>Can even be used 2 years after expire date.</td></tr><tr><td>Immobiline DryStrip Reswelling Tray</td><td>GE Healthcare</td><td>80-6371-84</td><td>Do not clean with organic solvents. Designed for 7-18 cm IPG-strips.</td></tr><tr><td>Immobiline DryStrip Kit</td><td>GE Healthcare</td><td>18-1004-30</td><td>Includes a tray, electrode holder, anode and cathode electrodes, aligner and sample cup bar and sample cups.</td></tr><tr><td>EPS 3501 XL Power Supply</td><td>GE Healthcare</td><td>18-1130-05</td><td>Supplies voltage up to 3500 V.</td></tr><tr><td>Multiphor II Electrophoresis Unit</td><td>GE Healthcare</td><td>18-1018-06</td><td>Movable electrodes enable IEF in IPG strips of all length (7-24 cm IPG strips)</td></tr><tr><td>PerfectBlue gel system Twin S</td><td>Peqlab</td><td>45-1010-C</td><td>SDS-PAGE in 10x10 cm mini-gel format. Gel chamber includes a cooling system.</td></tr><tr><td>staining dishes with lids</td><td>VWR international</td><td>216-3412</td><td>Fits for mini-gels. Stackable on shaker.</td></tr><tr><td>aluminium sulfate-18-hydrate </td><td>Merck &amp;amp; Co., Inc.</td><td>1.01102.5000</td><td>We made best experience with Merck. Just available in 5 kg package. </td></tr><tr><td>orthophosphoric acid</td><td>Prolabo</td><td>20 624.295</td><td>Sold in glass bottles.</td></tr></tbody></table>

fast and sensitive colloidal coomassie g-250 staining for proteins in polyacrylamide gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

Watch this Scientific Journal Video about Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels at JoVE.com

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

fast-sensitive-colloidal-coomassie-g-250-staining-for-proteins

Research

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Biology

40.3K Views.  University Duesseldorf. This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Video: Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Cancer Research

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

Genetics

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Biochemistry

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight

Source: Zhan, X., et al. <a href="https://www.jove.com/v/56739">Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome</a> J. Vis. Exp. (2018)
In this video, we demonstrate a two-dimensional gel electrophoresis method that separates individual proteins from a complex protein mixture obtained from tumor tissues of human nonfunctional pituitary adenoma (NFPA). The proteins are separated based on their electric charge in the first dimension and further separated based on molecular weights in the second dimension.

Dwuwymiarowa elektroforeza w żelu poliakrylamidowym: technika rozdzielania do analizy białek w złożonej mieszaninie białek na podstawie ładunku i masy cząsteczkowej

Source: Zhan, X., et al. Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Protein Analysis: A Technique to Separate and Visualize Proteins Based on Molecular Weight

Prepare two separating gels as described in the text manuscript. Pour the gel between the glass plates using a 1-milliliter pipette, ensuring that the upper 2 centimeters are free of the mixture. Add 70% ethanol on top of the separating gel, creating an even interface between the two layers.
Once the separating gels have polymerized, prepare the stacking gels using instructions in the text manuscript. Then, remove the ethanol from the separating gels, and add the stacking gel solution. Carefully insert a comb with the desired number of pockets, without introducing air bubbles, and allow the gel to polymerize for 20 to 30 minutes.
Load 4 microliters of each sample, as well as the protein ladder, into separate wells. Then, run the gel in Tris-Glycine running buffer at 144 volts for 45 minutes at room temperature. Use pre-warmed Fairbanks solution A to stain the gels on a rocker for 30 minutes, and pre-warmed Fairbanks solution D to decolor the gels on a rocker until the desired background is achieved.

Elektroforeza w żelu poliakryloamidowym z dodecylosiarczanem sodu do analizy białek: technika oddzielania i wizualizacji białek na podstawie masy cząsteczkowej