Diffusion Blotting Protocol - Creative Diagnostics

Introduction of Diffusion Blotting Protocol

Electrophoretic transfer of protein is the most widely applied method for western blotting after SDS-PAGE. When using precast gels supported on plastic films, removal of the film became necessary to allow electrophoretic transfer. Ultrathin gels (0.1–0.2 mm) are difficult to separate from the supporting material, and a method for diffusion blotting which gave efficient transfer was subsequently developed. Furthermore a method for diffusion blotting from 1.5 mm slab gels has been described. We wished to investigate diffusion blotting as an alternative method for the commercially available precast 0.5 mm SDS-PAGE Excel gels on plastic supports and obtain quantitative data concerning the efficiency of the method. The efficiency of diffusion blotting was compared with 1-h electroblotting, and it was found that the transfer rate was fast initially and that it declined with time. Compared to electroblotting, 10 % of the proteins was transferred during the first 3 min, 20 % was transferred after 10 min, and after 3 h 45–65 % was transferred. Diffusion blotting also allows for several blots to be made from one gel. A doubling of transfer time from each blot is necessary to obtain similar amount of proteins on each blot. After blotting, the proteins in the gel can be stained by standard methods such as Coomassie Brilliant Blue or silver staining. Diffusion blotting is a simple method which is performed with minimal equipment. The gel can remain on the solid matrix, which maintains the integrity of the gel and permits the generation of multiple blots. Several antisera can thus be tested on identical runs and these can be compared to protein staining of the same gel. The many advantages of diffusion blotting make this an alternative to electroblotting when ultimate sensitivity is not required.

Figure 1. Multiple blots of the same lane.

Materials of Diffusion Blotting Protocol

Prestained molecular mass standard from any supplier.
Nitrocellulose membrane.
3MM Chr paper.
Tris/glycine transblot buffer (pH 8.0): 25 mM Tris, 190 mM glycine, and 20 % (v/v) methanol. Adjustment of pH is not required.
Glass plate.
Weight.

Methods of Diffusion Blotting Protocol

After SDS-PAGE put the gel on a flat surface leaving the solid support in place (for blotting after 2D gel electrophoresis).
Use gloves when handling membrane. Cut appropriate number of nitrocellulose membranes of the exact same size as the gel. Cut upper right corner corresponding to the cut corner on the precast SDS-PAGE Excel gels to help correct orientation.
Soak the nitrocellulose membrane in the transblot buffer until wet.
Remove excess buffer by holding two corners of the membrane with your fingers and carefully allowing the edge to touch a sheet of filter paper.
Carefully lay the membrane onto the gel. Avoid air bubbles. Do not remove the membrane after it has been in contact with the gel because some protein is transferred immediately and this may result in double bands.
Wet three sheets of 3MM paper in transblot buffer. Remove excess buffer by squeezing the sheets between two fingers and place the papers on top of the membrane.
Cover the gel, membrane, and filter paper assembly with a glass plate and a weight of approximately 1.5 kg (6 g/cm ²).
Leave for 3 min.
Repeat steps 3 – 6 for every blot to be made and double the transfer time for each blot to obtain the same amount of protein on each blot.
Cut a mark in the nitrocellulose for each band in the prestained standard.

Notes of Diffusion Blotting Protocol

Instead of transblot buffer we have often used PBS pH 7.4 and it appears to work equally well.
The method is described for precast 0.5 mm SDS-PAGE gels. These gels are also used in a two-dimensional electrophoresis system where Immobiline DryStrip for isoelectrofocusing is used in the first dimension and the precast gels are used for the second dimension. The method works equally well following two-dimensional gel electrophoresis, where exact pattern reproducibility is difficult to achieve. Creation of multiple identical imprints by diffusion blotting will eliminate problems with gel-to-gel variation and facilitate correct identification of protein spots as well as easy comparison of different antisera.
The time can be changed according to the sensitivity of the detection system. Three minutes will give a transfer of approximately 10 % of what you see for electroblotting while 30 % is transferred after 30 min.
The prestained standard is visible after blotting, but will weaken considerably after blocking and development. Marking the bands with a knife will help in locating the molecular mass markers. If protein staining of the gel is performed, molecular mass determination can be done on the corresponding band identified on the gel.

Reference