Protein transfer from a gel to a blotting membrane is crucial in the success of your Western blot. Fortunately, you have transfer options. Two techniques allow you to choose the best method for your experiment: wet-tank and semi-dry transfer.
Wet-Tank Transfer
If your proteins have a broad range of molecular weights, you can transfer them all at once with the wet-tank transfer method. Keep in mind that wet-transfer typically takes longer to complete than semi-dry.
Buffer Composition
While buffer composition can be adjusted to your transfer needs, start with Towbin transfer buffer (25 mM Tris, 192 mM Glycine, 20% methanol (v/v), pH 8.3) for wet-tank transfers. It aids in promoting protein binding to the membrane and provides a high buffering capacity.
Methanol
Methanol is useful in wet-tank transfers because it prevents the gel from swelling. Additionally, it can promote protein binding to the membrane. Methanol does have a few drawbacks, however. It may reduce gel pore sizes, change the protein charges, and cause protein precipitation. Depending on the molecular weight of your target, you can adjust methanol concentration in increments. When targeting small proteins ( 150 kDa) you can reduce methanol to 5% and add 0.05% of SDS. These adjustments in methanol and SDS concentration should be performed in small increments.
Gel Equilibration
To prevent interference during protein adsorption to the membrane, equilibrate the gel prior to mounting your transfer sandwich. This will help reduce the amount of sodium dodecyl sulfate (SDS) and additional buffer salts in the gel and thereby decrease the chance of disruption. Before preparing your transfer, simply incubate your gel in transfer buffer for 15 minutes to let equilibration take place.
Power Settings and Transfer Time
Wet-tank transfer utilizes high-intensity power settings to create a short transfer time. However, this means that the system can generate too much heat; therefore, measures should be taken to prevent the samples from overheating and becoming damaged. Chilled transfer buffer and an ice unit are recommended. A low voltage (30V) transfer can also be performed overnight to improve efficiency over a broader range of molecular weights.
Semi-Dry Transfer
Compared to wet-tank, semi-dry transfer methods are much quicker. The process, however, is less flexible.
Transfer Buffer
Very little transfer buffer is needed for semi-dry transfer methods. This reduces the system’s buffering capacity. In terms of transfer buffer types, Towbin is suitable in most cases. However, Bjerrum and Schafer-Nielson transfer buffer (48 mM Tris, 39 mM glycine, 20% Methanol (v/v), pH 9.2) is also recommended to optimize efficiency.
A discontinuous buffer system may be useful to your experiment as well. Semi-dry transfer is unique because it can employ different buffers for each set of filter papers in the transfer stack. For example, a Tris-CAPS-methanol buffer can be used on the anode side and a Tris-CAPS-SDS buffer on the cathode side (60 mM Tris, 40 mM CAPS, pH 9.6 with either 15% methanol or 0.1% SDS).
Electrode Contact
To ensure an efficient transfer, arrange complete contact between the two electrodes and the transfer stack. During stack preparation, keep the membrane and filter paper sheets trimmed to the gel dimensions, and remove all bubbles during assembly.
Gel Equilibration
Several electrophoresis components, such as SDS, increase the conductivity of the transfer buffer and consequently the amount of heat generated during the transfer. It is therefore suggested that residual components are removed by equilibrating the gel in transfer buffer prior to transfer.
Power Settings and Transfer Time
In contrast to wet-tank, semi-dry transfer methods utilize a lower buffering capacity and high heat to create a short (15–30 min.) transfer time. To avoid any overheating and transfer complications, a longer (30–60 min.) time with constant current will result in slower protein transfer but may diminish complications.
Find a diagram of the complete Western blot workflow here. And, for more helpful tips, read the Protein Electrotransfer Methods and the Odyssey® Infrared Imaging Systems technical note.
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The Best Western Blot Transfer Method
Determining the best Western blot transfer method for your experiment may take some trial and error. Luckily, we're here to support you and your team every step of the way. If you have questions about reagents, transfer methods, or Western blotting in general, then please don't hesitate to contact us today. We're always eager to lend a hand and provide the tools and techniques needed to optimize your lab's protocols.Powered by Froala Editor