Name: Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels (Video) | JoVE Journal
Uploaded: 2007-08-22T00:00:00.000+00:00
Duration: 22 min 45 s
Description: Learn western blotting using the Invitrogen NuPAGE system for protein analysis in transfected cells. This reliable method enables efficient protein separation, transfer, and detection for reproducible results.

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5 minutes

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/pubmed?term=((Gel+Migration+chart%3A+%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Gel Migration chart: .</a> , 
 
 <a target="_blank" href="http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.Par.99253.File.dat/O-063575-NuPage_fin.pdf">http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.Par.99253.File.dat/O-063575-NuPage_fin.pdf</a> Forthcoming.</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complete+western-blot+protocol+from+the+Invitrogen+website%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Complete western-blot protocol from the Invitrogen website.</a> , 
		
 <a target="_blank" href="https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=A2C5758BC7C3438B01378A0940376C8E">https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=A2C5758BC7C3438B01378A0940376C8E</a> Forthcoming.</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NuPAGE+technical+guide%5BTitle%5D)+AND+%22%22%5BJournal%5D)">NuPAGE technical guide.</a> , 
 
 <a target="_blank" href="http://tools.invitrogen.com/content/sfs/manuals/nupage_tech_man.pdf">http://tools.invitrogen.com/content/sfs/manuals/nupage_tech_man.pdf</a> Forthcoming.</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Instruction+Manual%2C+Mini+Trans-Blot+Cell%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Instruction Manual, Mini Trans-Blot Cell.</a> , 
 
 <a target="_blank" href="http://www.bio-rad.com/cmc_upload/0/000/013/280/M1703930E.pdf">http://www.bio-rad.com/cmc_upload/0/000/013/280/M1703930E.pdf</a> Forthcoming.</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ECL+Plus+Western+blotting+detection+reagents+instruction%5BTitle%5D)+AND+%22%22%5BJournal%5D)">ECL Plus Western blotting detection reagents instruction.</a> , 
 
 <a target="_blank" href="http://www5.gelifesciences.com/aptrix/upp00919.nsf/Content/139B41E53EDC6210C1256EB40044ACE3/$file/RPN2132PL_Rev_D_2006_web.pdf">http://www5.gelifesciences.com/aptrix/upp00919.nsf/Content/139B41E53EDC6210C1256EB40044ACE3/$file/RPN2132PL_Rev_D_2006_web.pdf</a> Forthcoming.</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimation+of+protein+molecular+weights+by+SDS+gel+electrophoresis%2C+GE+Healthcare+Website%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Estimation of protein molecular weights by SDS gel electrophoresis, GE Healthcare Website.</a> , 
 
 <a target="_blank" href="http://www1.gelifesciences.com/aptrix/upp00919.nsf/Content/elpho_applications~elpho_applications_1d_protein_analysis~elpho_sds_page~Elpho_1D_SDS+PAGE+2+Estimation+of+protein+molecular+weights+by+SDS+gel+electrophoresis+~3.+Procedure">http://www1.gelifesciences.com/aptrix/upp00919.nsf/Content/elpho_applications~elpho_applications_1d_protein_analysis~elpho_sds_page~Elpho_1D_SDS+PAGE+2+Estimation+of+protein+molecular+weights+by+SDS+gel+electrophoresis+~3.+Procedure</a> Forthcoming.</li>
<li>Yeromin, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+identification+of+the+CRAC+channel+by+altered+ion+selectivity+in+a+mutant+of+Orai%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Store-dependent+and+-independent+modes+regulating+CRAC+channel+activity+of+human+Orai1+and+Orai3%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">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/pubmed?term=((The+CRAC+channel+consists+of+a+tetramer+formed+by+Stim-induced+dimerization+of+Orai+dimers%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.</a> Nature. , Forthcoming.</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 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.

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 ...

Hello, my name is OB Pena. I work in the Michael Car Lab in the physiology and biophysics department of the University of California. And today I will show you to perform a western blot using the new page system from individual gene.This is a basic technique to analyze expression of protein to check the results of a precipitation experiments. So western blood techniques involve three basic steps, loading and running the gel, transferring the protein from the gel onto a nitrocellulose membrane and immuno probing the membrane. Today I will use this technique to analyze the expression of a protein In translated cells.So I prepare the sample so the protein sample are always responding in a sample buffer for X.This sample buffer contains dye to help you to load your waste and blood glycerol to help the sample to go in the bottom of the well. It contains also buffer and LDS. This is a detergent negative negatively charge that we could the protein and the protein with some migrate in the electric fields, accounting to the molecular weight and normal to the charge.You need also to add some reducing agents as DTT to break the ine bone. So the sample are heated 10 minutes at 70 degree to fully the natural the protein and quickly spin on. So now I will prepare the gel.So we use precast gel, so we need to cut packaging, take the gel, peel off the protection. So now I'm ready to answer the gel in the setup. So when the gel is incorrect orientation, so riding should be facing you and the open parts facing the central part of the cells.So first you need to close tightly to a leakage and you add some running buffer in the central parts. I know I will add the running buffer in the other parts, the outside parts, you need to put enough buffer to avoid the gel to warm up during the run. No, I will take on take off the C.Yeah, you have to go slowly and vertically so you're loading from the bottom and you're moving up slowly and just be sure to avoid the bubble.At the end I'm gonna be loading seven samples and I had three molecular weights. This would help me to cut the membrane after the transfer to block different pathways, different antibodies just to know where I can cut the membrane and where are my samples. So here you have the three standards and after transfer will cut in the middle of the standards the membrane.So here you will have one block bloating with one antibody and here is the seven parts that will be bloating with another antibody. Just make sure when you load the gel that you load all the well and that you load the J the well with almost the same volume to have a nice migration. Now I'm ready to run the J.So we connect the apparatus to the power supply.So it's only one way to put the lead switch so you can run the gel either at constant voltage or constant current. So we do that constant voltage and for the first part and we just set the voltage to around 60 70 volts. When I apply the current, you will see bubbles that appear that mean that you have a go good conductivity and you will see the proteins starting to migrate to towards the bottom of the gel towards the positive electrode cuts.Due to the a DS negative chart that code the protein for the first part of the gel, it is called stacking just to stack the protein in one single bonds it's a low IDE concentration and in the second step of the run I will increase the voltage to hundred and 75 volts. It has been one hour and a half. And judging by the position of the standards you, you can know where the your protein is gonna be according to the molecular weight and compared to the molecular weight of the different unborn of the standard that you can see.So another, the run is over and I will take the gel off of the cells pathogen in the transfer buffer. That is a code using a forceps or spatula. You separate the two parts of the precursion and take, you cut the part with the well I can take the gel carefully and put the gel in the buffer.No, We will perform the transfer. It's, which means that we will transfer the protein from the gel towards the membrane nitrocellulose membrane. And for this purpose we will use a bi bio rat systems.It's a liquid transfer. Other system exists like a semi dry transfer depending of what you have in your lab. So the different part of the system are the cube, the transfer system with this things to prepare your sandwiches.So the principle is the same for phys, it's horizontal electrolysis this time. So you will prepare a sandwich with two pad to maintain the sandwich very wet. Will put your gel your membrane and two wet man paper on each side and put the sandwich in the system and the protein.Once again, we migrate towards a positive electrode from the gel to the membrane. So you need to add your own transfer buffer in a dish. So you're chasing the bubble out of the pan.Can you place the first pad at the bottom of the sandwich? Take one whiteman paper, put in the buffer and let's the buffer getting by capillary and plus press whiteman people on top of the pad Once again, get rid of the bourbon. Now you press the gel And your kids were better part of the jail.So you take your natural membrane, you can either cut an angle or write something on your membrane to remember the orientation. As you can see, I write a number on the top right angle to remember the orientation of my membrane. No, I will we the membrane in the can place the membrane on top of the gel in the good orientation, adding a new Whiteman paper and chasing the bour.It's the important step in the transfer process is to avoid bubble. You are, it is very important. Do not have bubble between the membrane and the gel, otherwise the protein will not transfer.You add the last spell And you close this on which, And you insert sandridge in the system. The black part facing the black part of the systems. If you have two gel, you it repeat the same operation with the other one and you fill the trunk with the transfer buffer.So now I will add the highest block for the transfer, close the system and transfer my protein for one hour at a hundred volts in the code. Can I also transfer your protein overnight at 20 volts? So now it has been waterer and the transfer is over.So we'll move to the next steps which immuno probing of the membrane. So first thing is to take your membrane out of the sandwich. So you placed the membrane in the, in a cream tree as you can see, know the standards are on the membrane and not on your gel anymore.To check the transfer efficiency I will perform a staining. It's a reversible protein staining and you runs sustaining with water. So you can see no the proteins standing red and that's transfer is correct.So now you had the blocking solution, so either PBS or TBS buffer with 5%milk or caffeine or B 2%BSA. It's purpose of that. It's to cover the membrane with proteins and to block all non-specific sites.So when in the next step you will put your antibody will not non-specifically bind into your membrane. And we leave the membrane like this at least 30 minutes at temperature. After one hour in the blocking solution, the membrane is ready to be probed by the, by the primary antibody.So you dumped in the sink your blocking solution and you add on your membrane the primary antibody solution. The primary antibody is diluted in the blocking solution also and you incubate your membrane for one or two hours at room temperature or overnight at four degrees Celsius. After the incubation with the primary antibody, you wash your membrane three times 10 minutes with PBS or TBS CONT containing 0.05%between 20.So if you d your solution in the sink and you add your washing solution or if you want, you can keep your antibody solution at four degree or minus 20 degree. After the three wishes, you incubate your membrane with a ary antibody coupled to the redish peroxidase for one hour at temperature. So in our case we are, we incubated the membrane with a primary antibody directly compared to HRP.So we, we just make the three rashes and reveal the waste. The Wahe are no hover and the membrane is ready to be revealed. So there is different way to detect your protein on the western blood membrane.Today we will use a Kim luminescence assay from you take your membrane, you dry it on a wet man paper, you put it in a clean tray or, and you prepare the substrate solution And you apply your solution on your membrane for five minutes. One or two ml are enough for mini gen. Five minute have passed.No, we're ready to expose the membrane. So you take the membrane, you dry it on the white man paper and you place it in a plastic polish wash or a serum wrap in western blood cassette you drain. So liquid is observing paper you see and you go in the dark room to expose a film on your membrane.We are in the dark room. No, I'm going to expose a film on the membrane for one minute. This step need to be performed in the dark.So you will just see the results at the end to a nice one on your film. You can change the exposure time. I'm introducing the film in the developer.So we Just got our film out of the developer. And to know exactly the size of the band that you can see on your film, you need to position the film on your membrane and to write on your film the position of the protein standards. In order to position the film, you align the, the fluorescent sticker on the marks on the field and you mark the standards.In this particular experiment, I've transfected htt cells with increasing amount of DNA. Here, here you have the line correspondence on non-infected cells and here, so increasing amount of DNA that correspond to an increasing amount of protein expressed. Here you can also observe non-specific bone.It's just a background of the antibody because it's also present in nonspecific non-specific cells. After you have phi, you can take your membrane, wash it in PBST. At this point you can keep your membrane and repeat the immuno immunostaining procedure with another antibody.After stripping your membrane, you can also catch your membrane in different pieces to, to use different antibodies At the same time, you can store your membrane at four degrees. Use in PBST or blocking Solution, I just show you To perform a western blood. As you saw my, my const is expressed in myself.So I wish you the same luck with your Experiments.

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

Research

JoVE Journal

Biology

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.

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

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

Western Blotting: Sample Preparation to Detection

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°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.

Pouring and Running a Protein Gel by reusing Commercial Cassettes

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Multimer-PAGE for Separating Native Protein Complexes: A Hybrid Separation Technique Consisting of Blue Native-PAGE and SDS-PAGE to Separate Intact Multimeric Proteins From Tissue Lysate

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

This protocol explores the latest advancements in performing Western blot analyses. These novel modifications employ a Bis-Tris gel system with a 35 min electrophoresis run time, a 7 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 Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

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.

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

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.

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

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.

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

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.

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Learn western blotting using the Invitrogen NuPAGE system for protein analysis in transfected cells. This reliable method enables efficient protein separation, transfer, and detection for reproducible results.

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

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