If your protein has a molecular weight greater than 150KD but is struggling to produce a visually appealing Western blot, then you must read this article.
It's important to choose the right gel
Three common gel include Tris Glycine, The PH of Bis Tris and Tris Acquire. Tris Glycine gel is 8.6, and its shelf life is very short. During the process of running the glue, the pH also showed an upward trend, reaching 9.5. This can lead to protein degradation and low resolution.
The PH of Bis Tris gel is 6.4. Compared with Tris Glycine gel, its stability and shelf life have increased. But this kind of gel needs to add antioxidant, such as DTT, in the solution to maintain the reducibility of protein.
The PH of Tris Acetate gel is 7, and the macromolecular protein will present a high resolution. When running macromolecular proteins, we recommend using Tris Acetate gel.
Gel concentration is important
Since Tris Acetate gel has been selected, there are still several issues to consider. The concentration of gel is inversely proportional to the pore size: the smaller the concentration, the larger the pore size. Larger proteins easily pass through larger pores, so I recommend using a low percentage of gel, such as 7%. Gradient gel or non gradient gel can be used, and gradient gel is suitable for new samples.
Increase SDS in transfer buffer and reduce methanol
The composition of the transfer buffer is crucial. If methanol is present, macromolecular proteins are easily precipitated. To avoid this situation, reduce the percentage of methanol in the transfer buffer (10% or lower). To further ensure that the protein does not precipitate, consider adding SDS to a final concentration of 0.1%. SDS adds uniform negative charges to the proteins, making them easy to transfer from gel to the membrane.
Choose the appropriate membrane
The membrane generally includes PVDF membrane or NC membrane. Choose PVDF membrane for running large molecule proteins. As mentioned earlier, if methanol is present, macromolecular proteins are easily precipitated. PVDF membrane does not require the addition of methanol in the transfer buffer, which increases the chance of target protein transfer. Both types of membranes have various pore sizes, commonly 0.2um and 0.45um. As with gel, larger proteins are easier to penetrate larger pores than smaller ones. Most proteins (>20kDa) can be transferred using a 0.45um membrane, avoiding the use of a 0.2um pore size.
Increase transfer time
After the correct selection of gel, transfer film and transfer buffer, transfer begins. Semi dry conversion is fast and convenient, but not suitable for large molecular proteins. I suggest wet transfer because the transfer time of large molecular proteins is very slow. I recommend transferring at 350-400 mA for 90 minutes or at 4 ° C, 40 mA, and transferring overnight.
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