Of the various Western blot methods, (immunoblotting) wet transfer is one of the most common.
What is Western blotting?
Broadly, Western blotting (immunoblotting, or protein blotting) is a technique used to detect specific protein molecules from a complex mixture of proteins. Usually this is a 7-step process. Click here for information on the Seven Steps to Western Blotting Guide...
When analysing Western blot transfer, the term ‘blotting’, also known as “protein transfer” or simply “transfer”, is the biological movement of samples from a gel to a membrane. Blotting fixes protein molecules in position ready for antibody labeling. Your colleagues might use the term “electrotransfer Western blot,” which involves applying a current that transfers the proteins off the gel and onto a stable membrane. To explore this in video, watch our Step by Step Western Blot video and our Western Blotting for Beginners video.
Getting your Western blot membrane transfer right the first time helps you improve laboratory efficiency and save time. In this post, we provide a simple Western blotting transfer guide for setting up a widely-used variation of protein blotting: wet transfer.
When should I use Western blot protein wet transfer?
You might be aware of various protein transfer methods, each with different reagent requirements, but wet transfer and semi-dry western transfers are the most common.
So how do you choose between wet transfer and semi-dry transfer for your Western blots?
Several factors influence the choice Western blotting experts must make between wet transfer and semi-dry transfer such as size of protein molecule, required speed and equipment available in the laboratory. Semi-dry transfer is fast and consumes less blotting reagents than wet transfer, but is more effective for smaller protein molecules. Larger protein molecules might not transfer effectively with the Western blot semi-dry transfer method. To explore polyacrylamide gel electrophoresis and western blots in more detail, read our article on molecular weight markers.
Wet transfer, on the other hand, can transfer all sizes of protein molecules, but is slower and consumes more reagents than semi-dry Western transfer. So, if time and reagents are not limiting factors for you, wet transfer is a good option for any Western blot transfer experiment.
Selecting a blotting protein transfer unit
One of the first things to consider when planning Western blotting protein transfer is the choice of transfer unit. The unit is the casing for the gel and the membrane that connects to an adjustable power supply to control the speed of the protein transfer.
If you don’t run Western blotting gel transfer often and you only need to run small (9 × 10 cm) gels, an integrated system for electrophoresis and electrotransfer is an attractive solution. This type of system combines two processes into one instrument, making for convenient and fast Western blotting, freeing up your time.
For larger Western blotting gel transfer—or for running many gels simultaneously—tank transfer units, such as TE22 and TE62, are more suitable. These units allow high throughput by running up to four large gels or 16 small gels at the same time. They also have built-in cooling systems to prevent protein molecule degradation.
Molecule membranes and blotting transfer buffers
Once you have chosen your Western transfer equipment, it’s time to select an appropriate membrane and blotting transfer buffer. Your choice of membrane affects the binding efficiency of your protein molecules, and the level of unwanted nonspecific background.
Find out more about immunoblotting membrane selection.
When setting up a Western blot transfer, ensure the membrane is in direct contact with the Western blot gel transfer on the side facing the anode (+). This means that the negatively charged protein molecules transfer from the gel to the membrane.
When it comes to immunoblotting transfer buffer selection, concentration of methanol and sodium dodecyl sulfate (SDS) are key parameters for Western blot transfer efficiency. These reagents affect the solubility of the protein molecule as well as its ability to bind to the molecule membrane.
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 0.1% SDS to support solubility and transfer speed.
In most cases, the optimal concentration of methanol in the Western transfer buffer is 20%. But, when you’re using low amounts of SDS to improve protein molecule solubility, you might need to reduce the concentration of methanol to as low as 10%. This will improve the ability of SDS to solubilize the protein.
Setting Western transfer time and current
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. This Western blot process and effect is known as “blow-through”.
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.
For more general information about effective protein molecule transfer, check out the Western blotting handbook.