This work was supported by grants from the National Institutes of Health and the George E. Hewitt Foundation fellowship (A.P.).

Gel electrophoresis
Technical note: Before starting the procedure, have your protein samples ready.
During this first step, the proteins in the sample are separated according to their molecular weight using denaturing polyacrylamide gel electrophoresis (PAGE). The NuPAGE&reg; LDS Sample Buffer loaded with Lithium Dodecyl Sulfate (LDS) maintains polypeptides in a denatured state once the protein sample has been heated at 70&deg;C for 10 minutes. A strong reducing agent is used in conjunction to remove secondary and tertiary structure (DTT, to break disulfide bonds). In addition, sample proteins become covered in the negatively charged LDS and therefore move through the acrylamide mesh of the gel toward the positively charged electrode. This allows their separation according to molecular weight (measured in kilo Daltons, kDa).
Detailed step-by-step protocols for the sample preparation and the PAGE procedure can be found on the Invitrogen website2, or in the NuPAGE technical guide3.
Technical tips
<ol>
<li>In the Invitrogen NuPAGE Bis-Tris discontinous buffer system, the electrophoretic mobility of the proteins and the subsequent separation range of the gel is dependent on two factors: i) the acrylamid concentration of the gel (with greater acrylamide concentration resulting in better resolution of lower molecular weight proteins) and ii) the trailing ion of the running buffer, MOPS or MES. To choose the appropriate combination for the separation range that you wish to achieve, refer to the Gel Migration chart1. The thickness of the gel and number of wells will depend on the volume and the quantity of samples that you plan to load, respectively.</li>
<li>When you load your samples into wells of the gel, at least one lane is reserved for a molecular weight marker (or ladder). These protein standards are commercially available from several companies and typically consist of a mixture of stained proteins having defined molecular weights, so as to form visible bands that allow you to follow the migration progress. Pick a range of molecular weights that is compatible with your gel resolution.</li>
<li>To achieve a nice and even migration pattern we recommend loading ALL the wells of your gel with a similar volume of sample or 1X LDS sample buffer.</li>
</ol>
Transfer
Technical Note: Before beginning the transfer step, prepare the transfer buffer (1X with 10% methanol) and pre-cool to 4&deg;C in a cold room.
In order to make the proteins accessible to antibody detection, they are transfered by electroblotting from the gel onto a nitrocellulose membrane. Protein binding is based upon hydrophobic interactions, as well as charge interactions between the membrane and protein.
(Polyvinylidene fluoride (PVDF) membrane can also be used as an alternative. In this case, the PVDF membrane needs to be pre-wet in methanol at least 30 seconds before use.)
A detailed step-by-step protocol of the transfer procedure can be found in reference 4 if you use the Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell, or in references 2-3 if your lab is equipped with the Invitrogen&rsquo;s XCell II&trade; Blot Module.
As a result of this process, the proteins are exposed on a thin surface layer and ready for detection. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by the reversible Ponceau S dye membrane staining.
Ponceau S staining procedure (optional):
<ol>
<li>After transferring the proteins, place the membrane in an incubation tray (proteins facing up).</li>
<li>Add enough Ponceau S Staining to cover the membrane and incubate at least 30 seconds with gentle agitation.</li>
<li>Rinse membrane with distilled water until the background is clear.</li>
<li>Destain the membrane with running distilled water for 2-3 minutes.</li>
<li>Place the membrane in blocking solution (see below).</li>
</ol>
Technical tips:
<ol>
<li>We recommend labeling your membrane with a pencil before protein transfer in order to denote the side in which the proteins were exposed and the orientation of your samples.</li>
<li>Both Ponceau S staining and the molecular weight markers may be used to cut the membrane after transfer for probing the resulting parts with different antibodies </li>
</ol>
Immunodetection:
Technical Note: This immunodetection procedure is provided as a guideline only. Optimization may be required for each antibody; specific information can be found in most data-sheets of the commercially available antibodies (e.g., working dilution, incubation time, etc.).
During this last process, the target protein will be detected using a specific antibody and will appear as a band on the film. The position of the band is dependent of the molecular weight of the target protein, whereas the band intensity depends on the amount of target protein present.
Typically, this is achieved after three substeps:
<ol>
<li>Blocking: To avoid non-specific interactions of the antibody with the membrane (the excess space on the membrane is covered with a dilute solution of a generic protein).</li>
<li>Probing: The protein of interest is detected by a specific <a title="Primary antibody" href="http://en.wikipedia.org/wiki/Primary_antibody">primary antibody</a>. After the unbound primary antibody is washed away, the membrane is exposed to a different antibody linked to the reporter <a title="Enzyme" href="http://en.wikipedia.org/wiki/Enzyme">enzyme</a>, <a title="Horseradish peroxidase" href="http://en.wikipedia.org/wiki/Horseradish_peroxidase">horseradish peroxidase</a> (HRP). This secondary antibody is directed against the species-specific portion of the primary antibody (e.g. an anti-rabbit secondary antibody will bind to any rabbit-sourced primary antibody). </li>
<li>Detection: A chemiluminescent agent is used as a substrate that will luminesce when exposed to the HRP on the secondary antibody. This reaction produces luminescence in place and in proportion to the amount of probed protein. The light is then detected by photographic film.</li>
</ol>
A generic step-by-step procedure is provided below. Other detailed procedures can be found in the ECL Plus Western Blotting Detection Reagents instruction manual5 or in most data-sheet accompanying the commercially available antibodies. Incubation time, antibody dilution, and blocking and wash solutions have to be empirically optimized for each antibody.
Immunodetection procedure:
<ol>
<li>Block the non-specific binding by incubating with enough volume of PBS 1% casein blocking solution to cover the entire membrane. Place on a rocker for at least 30 min at room temperature or overnight at 4&deg;C. </li>
<li>Incubate the membrane with your primary antibody (specific for the protein of interest). The primary antibody should be diluted in blocking solution. For most antibodies, complete binding is achieved after 1-2 hours incubation at room temperature under gentle agitation. Alternatively, the incubation step can be performed overnight at 4&deg;C to increase the signal to noise ratio. </li>
<li>Remove the solution (discard or keep at 4&deg;C to -20&deg;C for repeated use) and quickly wash the membrane once. Then 3X10 minutes with PBS, 0.05% Tween 20 (PBST) at room temperature with vigorous shaking.</li>
<li>Incubate the membrane with a HRP-coupled secondary antibody diluted in blocking solution. Choose an antibody which recognizes the IgG portion of the species where the primary antibody was raised. The incubation is performed for 1 hour at room temperature under gentle agitation.</li>
<li>Discard the solution and quickly wash the membrane once. Then 3X10 minutes with PBST at room temperature with vigorous shaking.</li>
<li>Proceed to the chemiluminescent detection according to the manufacturer instruction. We use the ECL Plus Western Blotting Detection Reagents from GE Healthcare (see reference 5 for specific instructions).</li>
<li>Place your membrane in saran wrap or a pouch and place inside an autoradiography cassette. Make sure to drain any excess liquid and remove all air bubbles.</li>
<li>Expose the photographic film to membrane in a dark room. Start with a 1 minute exposure time and adjust according to desired signal intensity.</li>
</ol>
Technical tips:
<ol>
<li>Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein. We typically use 1% casein, but <a title="Bovine serum albumin" href="http://en.wikipedia.org/wiki/Bovine_serum_albumin">bovine serum albumin</a> (~2% BSA) or non-fat dry milk (~5%) can be used as an alternative.</li>
<li>Tris-Buffered Saline (TBS) can be used throughout the procedure instead of PBS. We recommend using TBS if you plan to probe your membrane with phosphospecific antibody.</li>
<li>The use of a glow-in-the-dark sticker (e.g., Stratagene&rsquo;s Glogos&reg; II Autorad Markers) tapped in the bottom of the autoradiography cassette will help you to align your film and your membrane during the analysis step.</li>
</ol>
Analysis
Now that you have exposed your film, you will realize that in practical terms, not all Westerns reveal protein as one nice single band. Additional bands may also appear due to the non-specific binding of both primary and secondary antibodies. This background signal can be reduced by optimizing the immunodetection procedure. In addition, an appropriate control (e.g., untransfected cells, siRNA-treated cells, etc ) will be useful to determine the specificity of your antibody and the exact location of the target protein on the membrane.
After marking on the film the position of the stained protein standard bands from the membrane, plot the log of each molecular weight of the protein standards (y-axis) against their corresponding relative mobility (x-axis). Relative mobility (Rf) is the term used for the ratio of the distance the protein has moved from its point of origin (top of the gel) relative to the distance the tracking dye or a low molecular weight marker has moved (the gel front). Determine the regression line of the standard curve to obtain values for slope and y-intercept. The unknown molecular weight (size) of your target protein is estimated using its Rf and the following modified equation:
log molecular weight = (slope)(mobility or Rf of the target protein) + y-intercept
(see reference 6 for detailed instructions).
Expression level approximations are taken by comparing the band intensity of the target protein to that of a structural protein (e.g., tubulin or actin) or a housekeeping gene product such as GAPDH. This so called "loading control" should not change between samples and is revealed using a specific primary antibody. The image may be further analyzed by densitometry to evaluate the relative amount of protein staining and quantify the results in terms of optical density.

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+Migration+chart:+.">Gel Migration chart: .</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+western-blot+protocol+from+the+Invitrogen+website.">Complete western-blot protocol from the Invitrogen website.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NuPAGE+technical+guide.">NuPAGE technical guide.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+Manual,+Mini+Trans-Blot+Cell.">Instruction Manual, Mini Trans-Blot Cell.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECL+Plus+Western+blotting+detection+reagents+instruction.">ECL Plus Western blotting detection reagents instruction.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+protein+molecular+weights+by+SDS+gel+electrophoresis,+GE+Healthcare+Website.">Estimation of protein molecular weights by SDS gel electrophoresis, GE Healthcare Website.</a> , .</li><li>Yeromin, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+the+CRAC+channel+by+altered+ion+selectivity+in+a+mutant+of+Orai.">Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai.</a> Nature. 443, 226-229 (2006).</li><li>Zhang, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-dependent+and+-independent+modes+regulating+CRAC+channel+activity+of+human+Orai1+and+Orai3.">Store-dependent and -independent modes regulating CRAC channel activity of human Orai1 and Orai3.</a> J. Biol. Chem. 283, 17662-17671 (2008).</li><li>Penna, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRAC+channel+consists+of+a+tetramer+formed+by+Stim-induced+dimerization+of+Orai+dimers.">The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.</a> Nature. , .</li></ol>

The procedure presented here uses a Bis-Tris gel with MES running buffer as an example of denaturing PAGE using the Invitrogen NuPAGE Novex electrophoresis system. In addition, this pre-cast gel system allows protein separation under denaturing or non-denaturing conditions as well as accomodates a wide range of molecular weights (from 1-200 kDa for the Bis-Tris gels to 36-400 kDa for the Tris-Acetate gels). Depending on your experiment, you may choose to follow only part of the western-blotting technique described here a...

western blotting using the invitrogen nupage novex bis tris minigels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

Watch this Scientific Journal Video about Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels at JoVE.com

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

western-blotting-using-the-invitrogen-nupage-novex-bis-tris-minigels

Research

JoVE Journal

Biology

37.2K Views.  University of California, Irvine (UCI). This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen. 

Video: Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

Cancer Research

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Biochemistry

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Neuroscience

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 ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing conditions could be achieved in different plant species. Native protein:protein and protein:nucleic acid complexes from phloem samples have as yet scarcely been analyzed, although they might play important roles in maintenance of this specialized compartment or in long-distance signaling. Large molecular assemblies can be isolated using a blue native gel electrophoresis (BN-PAGE). Their protein components can be separated by a subsequent sodium dodecyl sulfate PAGE (SDS-PAGE). However, proteins with similar molecular weights co-migrate, what can hinder protein identification by mass spectrometry. Combining BN-PAGE with two different denaturing gel electrophoresis steps, namely Tris-Tricine-urea and SDS-PAGE, enables the additional separation of proteins according to their hydrophilicity/hydrophobicity and thus increases resolution and the success of protein identification. It even allows distinguishing proteins that only differ in their posttranslational modifications. In addition, blue native northern blotting can be applied to identify the RNA components in macromolecular complexes. We show that our protocol is suitable to unravel the protein and RNA components of native protein:protein and ribonucleoprotein (RNP) complexes occurring in phloem samples. Combining a blue native PAGE with two different denaturing PAGE steps can help to separate different kinds of large protein complexes, and also enables an increased identification rate of their components by mass spectrometry. Furthermore, the protocol is robust enough to simultaneously detect potentially bound nucleic acids within single protein complexes.

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing ...

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

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Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

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

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Pouring and Running a Protein Gel by reusing Commercial Cassettes

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes