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's definitely a beneficial staining protocol compared to other Coomassie protocols and even silver and fluorescent stainings.
Focus I: detection limit
In Kang's publication, they detected several marker proteins up to 1 ng/band (figure 2). But in our opinion a detection limit of 4-8 ng/band is more likely related to practice (table 1). They further discussed superior sensitivity levels in relation to both a colloidal Coomassie protocol with CBB G-250 in phosphoric acid, ammonium sulfate and methanol, and an acidic silver staining (Silver Stain Plus Kit) from Amersham Pharmacia Biotechnology.
<img src="/files/ftp_upload/1431/fig2.jpg" alt="Figure 2" /> Figure 2: Representative SDS-gel published by Kang et al. that demonstrates the power of the modified CBB-G250 protocol. 250 ng/band proteins were loaded at the most left and 2-fold diluted to the right side consecutively.
Table 1: Staining limits for standard proteins taken from Kang's publication in comparison to a more realistic threshold for protein detection.
<table border="1">
<tbody>
<tr>
<td>Protein</td>
<td>MW [kDa]</td>
<td>Protein amount [ng] (virtual vs. serious)</td>
</tr>
<tr>
<td>myosin</td>
<td>220</td>
<td>1 / 4</td>
</tr>
<tr>
<td>b-galactosidase</td>
<td>116</td>
<td>1 / 4</td>
</tr>
<tr>
<td>phosphorylase b</td>
<td>97</td>
<td>1 / 4</td>
</tr>
<tr>
<td>bovine serum albumin</td>
<td>66</td>
<td>2 / 4</td>
</tr>
<tr>
<td>ovalbumin</td>
<td>45</td>
<td>4 / 8</td>
</tr>
</tbody>
</table>
To confirm these detection limits and to further validate Kang&#x2019;s staining method we performed our own test series on common Laemmli SDS-gels with molecular weight markers detected by commercially available fluorescent staining solutions like Sypro Ruby (BioRad) and Deep Purple (GE Healthcare) (figure 3). Interestingly, Kang&#x2019;s staining scores well or even performs better (in case of Deep Purple) and is therefore definitely superior in terms of labor input and performance!
<img src="/files/ftp_upload/1431/fig3.jpg" alt="Figure 3" /> Figure 3: Comparison of Kang's CBB-G250-based staining (A) with Sypro Ruby (B) and Deep Purple (GE Healthcare) (figure 3). (C) staining of protein bands in SDS-PAGE. A molecular weight marker containing phosphorylase b (97.4 kDa), BSA (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa) was analysed, with protein amounts of 1 g/band at the left side of the gel and consecutively diluted 2-fold to the right.
Nevertheless, these detection limits were determined with standard proteins used for molecular weight markers and should be considered with caution. For this reason, we additionally tested Kang's staining method with different proteins that partly have weak binding affinity for CBB (e.g. fetuin, mycin). As demonstrated in figure 4, Kang's protocol is superior to the conventional CBB staining and even performs better than Sypro Ruby, but does not come up to silver staining in this case. However, we believe Kang's modified staining method is an outstanding alternative for sensitive and fast protein detections in gel-based proteomics.
<img src="/files/ftp_upload/1431/fig4.jpg" alt="Figure 4" /> Figure 4: Protein staining of fetuin with (A) Kang's colloidal Coomassie protocol (without destaining) compared to (B) a conventional CBB G-250 staining (40% methanol and 10% acetic acid), (C) Sypro Ryby and (D) silver staining according to Shevchenko et al. Protein amounts of 2 g diluted up to 2 ng were loaded for SDS-PAGE.
Focus II: usability Beside the improved detection limits does Kang's Coomassie protocol offer several features that makes it preferential towards the stainings of Neuhoff or Candiano and even fluorescence protocols:
<ul>
<li>fixing and staining is done in one step</li>
<li>not more than 4 steps in all</li>
<li>first staining results are fast visible (in most cases within less than 20 minutes)</li>
<li>the procedure requires only 2 hours for 80% completion of the staining</li>
<li>just minimal background staining</li>
<li>the staining procedure is very reproducible (it is an endpoint method)</li>
<li>no more handling with toxic alcohol (methanol is replaced by ethanol)</li>
<li>the solution is almost odourless (no use of acetic acid)</li>
<li>use of CBB G-250 in low concentrations.</li>
</ul>
In summary, the formula is quite convenient, relatively nonhazardous, environment friendly and cheap. Furthermore Kang's staining is completely compatible with mass spectrometry analyses. In the end one can say that Kang's Coomassie staining is excellent for a fast and analytical in-gel detection of proteins even for proteomics based approaches.

<table border="1" >
 <tr>
 <td>Name of product</td>
 <td>Company</td>
 <td>Catalogue No.</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>For IEF and SDS-PAGE:</td>
 <td></td>
	<td></td>
	<td></td>
 </tr>
 <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>For colloidal Coomassie G-250 staining:</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>staining dishes with lids</td>
 <td>VWR</td>
 <td>216-3412</td>
 <td>Fits for mini-gels. Stackable on shaker.</td>
 </tr>
 <tr>
 <td>aluminium sulfate-18-hydrate </td>
 <td>Merck</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>
</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

Nanyang Technological University

Research

JoVE Journal

Biology

40.2K 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

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 ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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 ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.