Buffers & Reagents Preparation For Western Blot
In western blot, except lysis buffer which is needed in sample preparation, other reagents also have to be prepared for western blot. Loading buffer, running buffer, coomassie brilliant blue staining solution, and coomassie destaining solution are needed to be prepared for SDS-PAGE, while western blot transfer buffer preparation is required for protein transfer. Sometimes, ponceau red staining is an alternative to check whether the protein transfer is successful, so a recipe of ponceau red staining solution is necessary. Besides, TBS buffer, blocking buffer, and TBST buffer are also needed to be prepared. The table below is a recipe especially about buffer or reagent needed in western blot, or we can name this table after “western blot buffer recipe”.
Comparison Of Blotting Membranes
When choosing a membrane, a protein’s properties and the downstream application will determine which membrane to use. Finding the optimal membrane may require experimenting with your specific protein on different membranes. Knowing the properties and the advantages and disadvantages to each membrane will help determine the best format for your application.
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Choose The Right Membrane
If you are transferring a low MW protein , use membranes with 0.2 µm pore size. For most other proteins, 0.45 µm pore size membranes can be used. Alternatively, PVDF membranes can be used.
PVDF is good for stripping and reprobing. For fluorescence western blotting, use low fluorescence PVDF . Remember that methanol activation is needed for PVDF membranes.
Nitrocellulose is a cost-effective option and no methanol activation is required with these membranes. Nitrocellulose membranes work well for IR detection.
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Tris Glycine Transfer Buffer Recipe
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Transfer Buffer For Western Blots
Since the first publication of a method for the electrophoretic transfer of proteins to nitrocellulose, the technique now called western blotting has become a standard method for detecting and quantifying proteins. The basic method of blotting and the composition of the standard western blot transfer buffer have not changed over the years.
Every western blot buffer must have two main properties: a western blot buffer must both promote the elution of proteins from the gel matrix and facilitate the efficient binding of all proteins in the sample to the membrane. Additionally, the components of a western blot buffer must not interfere with any subsequent steps after the transfer.
The advent of semi-dry blotting and the optimization of western blotting transfer for specific classes of proteins have resulted in several alternative formulations of western blot transfer buffers. Bio-Rad offers premixed blot buffers and reagents for use in common western blotting protocols.
Know Your Transfer Method
Understanding the different methods of transfer and which one would be optimal for your protein is the first step. Wet transfer, where the membrane sandwich is completely immersed in the transfer buffer in a buffer tank, is used for transferring proteins of broad molecular weights. In the semi-dry method of transfer, the buffer is restricted to the two stacks of filter paper covering the membrane. Semi-dry conditions are favorable for transferring proteins in the 30120 kD range. A third method, the rapid semi-dry transfer has special buffer and material formulations that facilitate the transfer of proteins in as short a time as in 310 minutes. The rapid semi-dry method can be used for proteins with a broad range of molecular weights.
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Western Blot Transfer Buffer Formulations
The standard transfer buffer for western blots, called Towbin buffer, is 25 mM Tris, 192 mM glycine, pH 8.3 usually with 20% methanol . Sometimes SDS is added to this buffer, generally in the range of 0.1 to 0.25%. This transfer buffer has both low ionic strength and low conductivity, which is optimal for tank blotting and for some semi-dry apparatuses.
Tris/glycine western blot buffer may not be suitable in some types of apparatuses for transfer of very high molecular weight proteins, which require lengthy transfer times. As transfer proceeds for an extended period of time, the production of heat decreases the resistance of standard western blot transfer buffer, causing the blot buffer to lose buffering capacity, thus reducing transfer efficiency. Additionally, the increased heat can cause gels to stick to the membrane, creating a handling problem for the soft, low-percentage acrylamide gels that are usually used for very high molecular weight proteins. Often, these gels must be carefully and laboriously scraped off the membrane.
For particular proteins, the choice of blot buffer can impact the efficacy of transfer. Generally acidic proteins are transferred more efficiently in a western blot buffer with a lower pH, and basic proteins are more efficiently transferred in a blot buffer with a higher pH. There are many buffers used for western blotting, such as the Dunn carbonate buffer 10 mM CAPS, pH 11 and 10 mM CHES, pH 9.6.
Loading And Running The Gel
The time and voltage may require optimization. We recommend following the manufacturers instructions. A reducing gel should be used unless non-reducing conditions are recommended on the antibody datasheet.
The gel percentage required is dependent on the size of your protein of interest:
|
Protein size |
Gradient gels can also be used.
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Transferring The Protein From The Gel To The Membrane
The membrane can be either nitrocellulose or PVDF. Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the stack. The time and voltage of transfer may require some optimization. We recommend following the manufacturers instructions. Transfer of proteins to the membrane can be checked using Ponceau S staining before the blocking step.
Prepare the stack as follows:
Figure 1. Example of prepared stack.
Small Electrophoretically Mobile Proteins
Useful notes.
Small electrophoretically mobile proteins will rapidly move out of the gel and actually through a protein binding membrane without prior precautions to retain them. There are generally four approaches taken to reduce losses due to trans-migration through the gel, these are 1) to reduce the methanol content which improves the hydrophobic nature of the surface of the protein, 2) reduce the time or transfer, 3) perform transfer in colder conditions, and 4) to dry the blot after electroblotting. These four modifications to the standard protocol are usually used altogether and allow 10-50 fold higher signal detection.
Two other general points are that SDS must be removed from the separating gel prior to electroblotting, failing to do this will cause small proteins to pass straight through the membrane because its charge has been modified to a higher pI than the pH of the buffer by the sulphate groups of SDS. This can be accomplished by simply equilibrating the gel in transfer buffer for 10 min with agitation prior to assembling the transfer setup. A great control sample for an phosphoryl western blot is a cell culture treated with a broad spectrum phosphatase inhibitor such as 100 micromolar sodium vanadate for 4 h pre-lysis.
Required materials and buffers
Required equipment
Standard liquid based polyacrylamide gel transfer system from Biorad or GE Healthcare or other supplier .
Protocol
Notes about the sandwich
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Comparison Of Western Blot Transfer Methods: Wet Semi
Efficient and reliable protein transfer from the gel to the blotting membrane is the cornerstone of a successful western detection experiment. Accuracy of results is dependent on the transfer efficiency of the western blotting method. Traditional wet transfer offers high efficiency, but at a cost of time and hands-on effort. Semi-dry blotting provides more convenience and time savings compared to traditional wet transfer, with flexibility to use multiple types of buffer systems or pre-assembled or build-it-yourself transfer stacks. However, semi-dry transfer can have a lower efficiency of transfer of large molecular weight proteins . Dry electroblotting offers both high quality transfer combined with speed as well as convenience since added buffers are not required for dry electroblotting.
| Cooling may be required for longer transfers | Multiple methods can be used including Towbin buffers | Requires pre-assembled transfer stacks |
Comparison of wet, semi-dry and dry transfer methods. A431 lysate was serially diluted on a Novex Tris-Glycine 4-20% gel. Proteins were transferred using the Mini Blot Module in the Mini Gel Tank, iBlot2 Transfer Device, Power Blotter, and the Bio-Rad TransBlot Turbo.
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Other Methods Of Transfer
Diffusion blotting
Diffusion blotting relies on the thermal motion of molecules, which causes them to move from an area of high concentration to an area of low concentration. In blotting methods, the transfer of molecules is dependent upon the diffusion of proteins out of a gel matrix and absorption to the transfer membrane. As the absorbed proteins are “removed” from solution, it helps maintain the concentration gradient that drives proteins towards the membrane. Originally developed for transferring proteins from IEF gels, diffusion blotting is also useful for other macromolecules, especially nucleic acids. Diffusion blotting is most useful when preparing multiple immunoblots from a single gel. Blots obtained by this method can also be used to identify proteins by mass spectrometry and analyze proteins by zymography. Protein recoveries are typically 25â50% of the total transferrable protein, which is lower than other transfer methods. Additionally, protein transfer is not quantitative. Diffusion blotting may be difficult for very large proteins in SDS-PAGE gels, but smaller proteins are typically easily transferred.
Vacuum blotting
The transfer efficiency of vacuum blotting varies within a range of 30 to 65%, with low molecular weight proteins at the high end of this efficiency range and high molecular weight proteins at the low end. Like diffusion blotting, vacuum blotting allows only a qualitative transfer.
Optimize Conditions When Switching From One Method To Another
Tris-HCl or Bis-Tris gels to TGX gels:
TGX gels afford fast transfers compared to other gels. However, in some cases proteins may blow through the membranes, resulting in loss of proteins. To avoid this, the following conditions are recommended:
Change only the transfer time and no other parameter. In general, you need only 50% of the time required for NuPAGE gels for proteins up to 250 kD. You might still need to tweak it.
TGX to Tris-HCl or Bis-Tris:
Double the transfer time .
Short transfer time to overnight transfer time:
For semi-dry transfers with the Trans-Blot SD semi-dry transfer system, start with 10 V for 30 min or 15 V for 15 min. For overnight transfers, a 30 V, 16-hr condition is recommended.
Tank to rapid transfer
Dont equilibrate the gel before transfer use it directly. If you are using PVDF membranes, be careful not to dry them out. Use the appropriate protocol, based on the MW of your protein, and then tweak the conditions.
For more details on different transfer methods, conditions, and troubleshooting tips, refer to Bio-Rads Protein Blotting Guide.
NuPAGE is a trademark of Invitrogen. SYPRO is a trademark of Life Technologies Corporation.
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Western Blot Protocols And Recipes
A western blot experiment, or western blotting, is a routine technique for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. Here, you can find a collection of western blot recipes for commonly used protein electrophoresis and western blot buffers and stock solutions, and general western blotting protocols for chemiluminescent and fluorescent detection to guide you through your experiment.
If you find this doesnt work for your specific protein of interest, try our BlotBuilder Product Selection Tool to get a set of recommended products with a personalized western blot protocol.
Role Of Sds And Methanol In Western Blot Transfer Buffers
For large proteins, the addition of SDS to the western blot transfer buffer can increase the efficiency of transfer, whereas for small proteins, particularly with nitrocellulose membranes having larger pores , SDS-denatured proteins may migrate faster through the membrane. The addition of SDS increases the ionic strength of the blot buffer and therefore increases heating additionally, depending on the apparatus and transfer conditions, the presence of SDS can lead to excessive foaming.
The addition of methanol to a western blot transfer buffer has been suggested to prevent gel swelling during transfer and improve the efficiency of protein adsorption onto the membrane. Many laboratories no longer use methanol since they have found that, for their protein samples, the addition of methanol does not markedly increase efficiency, and omitting it eliminates the problem of disposing of toxic methanol-containing solutions. To eliminate the problem of toxic waste disposal, some formulations replace 20% methanol with 10% ethanol.
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Types Of Western Blot Transfer Buffers
Transfer buffers must enable both effective elution of proteins from the gel matrix and binding of the protein to the membrane. While different gel types and blotting applications call for different transfer buffers, the choice of buffer also depends on the membrane being used and the physical characteristics of the protein of interest. This section provides an overview of different transfer buffers, such as Tris/Glycine, CAPS, Dunn carbonate buffers and discontinuous buffer systems. It also provides general guidelines for selection of buffers and membranes according to gel types and applications.
To maintain conductivity and pH transfer buffers contain a conductive, strong buffering agent . In addition, alcohol may be included in the transfer buffer to promote binding of proteins to membranes, and SDS may be added to promote elution of proteins from gels.
Regardless of the transfer buffer selected, when preparing and using transfer buffers:
- Do not use the same batch of transfer buffer more than once, as the buffer will likely lose its capacity to maintain a stable pH during transfer
- Do not dilute the transfer buffer this will also decrease buffering capacity
- Do not adjust the pH of transfer buffers when not indicated, as this increases buffer conductivity, which is manifested by higher initial current output and decreased resistance
General guidelines for transfer buffer and membrane selection by gel type.
| Gel Type |
Adjust Buffers Based On Your Protein
Whether you are using an existing lab protocol or one from a publication, you may need to recalibrate buffer formulations based on your protein of interest.
You can start with Towbin transfer buffer and alcohol as the basic buffer for any of the proteins and recalibrate based on how it performs.
Varying the amounts of SDS and Alcohol
Concentrations of methanol and SDS can be adjusted to improve transfer efficiency. Alcohol increases binding of SDS-bound proteins to nitrocellulose, but decreases pore sizes in the gel. Eliminating alcohol from SDS-protein transfers results in considerably diminished binding. Adding SDS to the transfer buffer increases the transfer efficiency of proteins, but reduces the amount of binding to the membrane. Therefore, if SDS is added to the transfer buffer, it is important to also include methanol .
SDS also increases the conductivity of the buffer and the heat generated during transfer. This might result in denaturation of some proteins. Large proteins denatured by SDS may transfer poorly if alcohol is added to the transfer buffer. Using a smaller pore size nitrocellulose membrane , can be effective in eliminating this loss.
If your protein has a high molecular weight or high pI, you can try the following buffer options:
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