Can you transfer a western blot too long?

If you are transferring for too long, it will also detect proteins in the second membrane. This is more likely to happen to smaller proteins first, so pay special attention to where the lower molecular weight ladder bands should appear. These three problems are very common. Most of them can be solved by hermetically packaging the transfer sandwich.

The transfer sandwich must apply equal and firm pressure to the entire stain. The efficiency of protein transfer will reflect any difference in pressure. The transfer of sandwiches with insufficient pressure between the gel and the membrane can also cause a loss of sharpness in the band pattern. You can minimize bubbling by degassing the buffers, preventing the creation of bubbles by pouring the tampon slowly by filling the tank and carefully assembling the transfer sandwich by pre-moistening the sandwich pads and using a roller at each step to expel the bubbles trapped in the sandwich.

The spotless method can be used to image gels before transfer and obtain images of the membrane after transfer, allowing direct quantification. Therefore, if SDS is added to the transfer buffer, it is important to also include methanol (10— 20%). Whichever transfer method you choose, the conditions must be optimized for the best transfer efficiency. Originally developed to transfer proteins from IEF gels (isoelectrically focused), diffusion transfer is also useful for other macromolecules, especially nucleic acids.

In transfer methods, the transfer of molecules depends on the diffusion of proteins out of a gel matrix and on absorption to the transfer membrane. Protein recoveries typically account for 25 to 50% of the total transferable protein, which is lower than other transfer methods. Diffusion transfer can be difficult for very large proteins in SDS-PAGE gels, but smaller proteins are usually easily transferred. The efficient and reliable transfer of proteins from the gel to the transfer membrane is the cornerstone of a successful Western detection experiment.

Diagram showing the assembly of a typical western transfer tank apparatus with the position of the gel, the transfer membrane and the direction of the protein in relation to the position of the electrode. The Invitrogen iBlot 2 dry transfer system provides a quick western transfer without the need for shock absorbers. PVDF membranes are highly hydrophobic and must be pre-moistened with methanol or ethanol before being immersed in the transfer buffer. In addition to the challenges of immunodetection in the protein transfer workflow, the transfer of proteins from a gel matrix to a membrane is a potential obstacle.

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. The iBlot 2 system performs comparable to traditional wet transfer methods in a fraction of the time. You can start with Towbin transfer buffer (25 mM Tris, 192 mM glycine, pH 8) and alcohol (20% methanol or 10% ethanol or 15% isopropyl alcohol) as a basic buffer for any of the proteins and recalibrate according to performance.

Leave Message

Required fields are marked *