Immobilizing proteins on a solid support is a first and crucial step in many protocols. The immobilized material can be used for detection of a single protein species using antibodies, radioactivity, etc. Nitrocellulose membrane provides the sticky surface where nucleic acids and proteins bind and it is widely used in various experimental protocols.
Bio-Dot, from Bio-Rad laboratories, is a simple device that offers a reproducible method for binding proteins or nucleic acids onto nitrocellulose or Zeta-Probe membrane. Bio-Dot has 96 wells that create 96 spots on the nitrocellulose. It is made of polysulfone plastic that can be autoclaved or washed with pure ethanol or sodium hydroxide (it is resistant to strong acid or base solutions), without destroying the device. Furthermore, the sample template attaches firmly to the gasket, thus minimizing any lateral leakage. As an additional precaution, however, it is always a good idea to leave empty lanes between your samples, if you have the space, of course.
The design of the sample template is similar to that of microtitre plates, allowing you to use your favorite multiple-tip pipette for loading the samples. You must bear in mind that nitrocellulose must be soaked in buffer before placing it between the gasket and the gasket support plate. To achieve perfect attachment, I would suggest to first soak the nitrocellulose in buffer, and then assemble the gasket support plate in the vacuum base and the gasket on top. After that, apply vacuum and then carefully place the nitrocellulose on the gasket. If the membrane attaches firmly onto the gasket, the vacuum will force it to stick firmly and it will suck away any buffer that has remained on the surface of the membrane. Next, carefully place the sample template on top of the nitrocellulose and finger tighten the screws in a diagonal crossing pattern to ensure even pressure all over the membrane surface. After loading each well, the vacuum will pull the solute away and the nucleic acids/proteins will attach on the nitrocellulose.
Sometimes, the vacuum fails to empty the solution out of some wells. This is due to small air-bubbles that form between the membrane and the sample template and prevent solute flow. A solution to this problem is to gently disrupt the bubble by placing a blue pipette tip in the well and leaving it there to gently touch the surface of the membrane. This trick has worked perfectly fine for me. Do not forget that you must be very careful not to tear the membrane. Using the tip gently should restore flow successfully.
The flow can be adjusted in three ways: full vacuum, gentle vacuum and atmospheric pressure. Thus, using the valve allows you to adjust the vacuum for passive filtration when binding samples or for rapid vacuum-assisted washes.
After the solute has been removed from each well, the proteins/nucleic acids are attached to the surface of the membrane. From this point, you can dry the membrane and use it for your downstream applications.
Graduate Student
Department of Biology
University of Crete