A great control sample for an phosphoryl western blot is a cell culture treated with a broad spectrum phosphatase inhibitor such as micromolar sodium vanadate for 4 h pre-lysis. Standard liquid based polyacrylamide gel transfer system from Biorad or GE Healthcare or other supplier we have found all the mini-gel blotting apparatus works well, but you should avoid rapid dry blotting as these are difficult to optimize and have in general high backgrounds.
Phosphorylated proteins gain a negative charge at the expense of a hydroxyl group usually from modified serine, tyrosine or threonine amino-acids which may shift the overall charge pI of the molecule to the acidic end of the pH scale. Another adaptation is to process samples rapidly on ice in the presence of phosphorylase and kinase inhibitors such that the level of phosphorylation is not affected by reverse hydrolysis of the phosphodiester bond which can be labile. Two other general points are that SDS must be removed from the separating gel prior to transfer for proteins less than kDal, failing to do this will allow the protein 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.
High molecular weight HMW proteins have low mobility through polyacrylamide gels which reduces transfer by reducing the amount of protein that can reach the membrane under standard transfer conditions. There are several techniques that can be used to optimize the transfer by increasing mobility. These are three fold; 1 add SDS to the transfer buffer, 2 increase the time for transfer, these modifications should be tried before the third method which needs more titration for optimization.
The optimal amount of SDS is 0. The SDS must be in the equilibrating buffer prior to assembling the transfer sandwich, use 10 min with agitation prior to assembling the transfer setup. A great control sample is to use nanograms of skeletal myosin as a well known large kDal protein. An example acid treatment could be 10, 30, or min in mM acetic acid, then equilibrating in transfer buffer prior to electroblotting.
SDS is not required for this process. Protease treatment of the gel can be with 0,, 0. Acetylated proteins gain a negative charge at the expense of a positive charge usually from modified lysine or arginine amino-acids which may shift the overall charge pI of the molecule to the acidic end of the pH scale. Another adaptation is to process samples rapidly on ice such that the level of acetylation is not affected by reverse hydrolysis of the acetyl group by deactylase enzymes or pH of the sample solution.
A great control sample for an acetyl western blot is a cell culture treated with a broad spectrum deacetylase inhibitor such as micromolar trichlorostatin A also called Trichostatin A for 4 h pre-lysis. Gels also have a tendency to adhere to the membrane after transfer, but rehydration of the gel can help facilitate separation.
Like diffusion blotting, vacuum blotting allows only a qualitative transfer. The most common immobilization membranes for western blotting are nitrocellulose, polyvinylidene difluoride PVDF , and nylon. These membranes are commonly used because they offer:. Most proteins can be successfully blotted using a 0. Nitrocellulose membranes are a popular matrix used in protein blotting because of their high protein-binding affinity, compatibility with a variety of detection methods, and the ability to immobilize proteins and glycoproteins.
Protein immobilization is thought to occur by hydrophobic interactions, and high salt and low methanol concentrations improve protein immobilization to the membrane during electrophoretic transfer, especially for proteins with higher molecular weights. Nitrocellulose membranes remain a popular choice due to the high efficiency of irreversible protein binding.
PVDF membranes have a high binding affinity for proteins and nucleic acids and may be used for applications such as western, southern, northern and dot blots. PVDF membranes are highly hydrophobic and must be pre-wetted with methanol or ethanol prior to submersion in transfer buffer.
In these applications, binding likely occurs via dipole and hydrophobic interactions. Due to the hydrophobicity of PVDF membranes, these are the preferred choice for hydrophobic proteins i. PVDF is less brittle and fragile than nitrocellulose and may be useful for western blotting experiments requiring multiple rounds of reprocessing stripping and reprobing procedures for different targets using a new combination of antibodies.
Charged nylon polyamide membranes bind proteins and nucleic acids by ionic, electrostatic, and hydrophobic interactions. The high durability of nylon membranes offers advantages in western blotting experiments requiring stripping and reprobing procedures. A significant drawback to using nylon membranes for blotting applications is the possibility of nonspecific binding and strong binding to anions like SDS. When choosing a membrane, a protein's properties i.
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. Explore: Transfer membranes. Several different transfer buffers are used for wet transfer methods. The type of buffer used is dependent on the protein of interest, the gel buffering system and transfer method. In most experiments, SDS is omitted from the western transfer buffer because the negative charge imparted to proteins can cause them to pass through the membrane.
It should be noted that adding SDS to the transfer buffer may require optimization of other transfer parameters e. Methanol is included in most transfer buffer formulations because methanol aids in stripping the SDS from proteins from separation by SDS-PAGE, increasing their ability to bind to support membranes.
However, methanol can inactivate enzymes required for downstream analyses, and it can shrink the gel and membrane, which may increase the transfer time of large molecular weight proteins kDa with poor solubility in methanol.
In the absence of methanol, though, protein gels may swell in low ionic strength buffers, and therefore it is recommended to pre-swell gels for 30 minutes to 1 hour to prevent band distortion. Explore: Transfer Buffers. Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Search All. Western Blotting Transfer Methods. See Navigation. Page contents Transfer methods Blotting membranes Transfer buffers. My Account. Browse Catalog.
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Sample Prep. Image Acquisition. Image Analysis. Better Transfer Efficient transfer of proteins from gel to membrane. Protein Blotting Workflow. Select Method and Equipment for Transfer Select from either traditional tank transfer, semi-dry, or advanced rapid semi-dry transfer methods. Prepare Transfer Buffer and Reagents Prepare transfer buffer sufficient for the transfer cell and for equilibration of gels and membranes. Note that rapid semi-dry systems use specialized buffers and may not need gel equilibration.
Equilibrate Gel and Membrane Equilibrate gels and membranes in transfer buffer if necessary. Assemble Gel and Membrane Sandwich Place gel and membrane between buffer-soaked filter papers. Perform Transfer Place transfer sandwich in transfer cell and fill with buffer.
Transfer Methods There are three main types of transfer apparatus and procedures: tank transfer systems, semi-dry systems, and rapid-transfer systems. Tank Transfer Systems These systems are useful for most routine protein work and for transfers of proteins of all sizes; gels and membranes are submerged under transfer buffer in tanks.
Semi-Dry Systems Gels and membranes are sandwiched between buffer-wetted filter papers that are in direct contact with flat-plate electrodes. Rapid-Transfer Systems A recent development, these systems utilize specialized apparatus that use proprietary filter papers and specialized buffers to rapidly and efficiently transfer proteins. Rapid-Transfer Systems. Transfer Buffers Transfer buffers contain a conductive, strong buffering agent for example, Tris, CAPS, or carbonate in order to maintain the conductivity and pH of the system during transfer.
Membrane Types Nitrocellulose and Supported Nitrocellulose Nitrocellulose membranes are easily wetted in water or transfer buffer, and it is compatible with a wide range of protein detection systems. Many Western blotting protocols remove SDS entirely from the blot transfer process by washing the gel in transfer buffer first.
The only exception is when transferring large or hydrophobic protein molecules. These proteins might require up to 0.
This will improve the ability of SDS to solubilize the protein. Two final parameters need to be set: current and Western blotting transfer time. These parameters help to create the optimal conditions for efficient and complete transfer of the protein bands on the molecule markers.
Insufficient current or Western transfer time can result in protein remaining on the gel. High transfer times or currents, on the other hand, can lead to smaller protein molecules with high electromobility passing right through the membrane.
High currents carry the extra risk of overheating the transfer buffer and the gels, which warm up in the blotting process anyway. Selecting the right Western blotting equipment and optimizing settings can improve laboratory efficiency and help you carry out successful Western blotting protein transfers.
Check out our Western blot blog post on optimizing protein transfer.
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