Semi-dry blotting

After electrophoresis, the gel is fixed and stained as other PAGE gels, too. For semi-dry blotting the 1 20 diluted buffer H is recommended. 

The semi-dry blotting apparatus consists of two plates (anode and cathode) made from graphite, glassy carbon, or stainless steel and a sandwich consisting of buffer-soaked filter paper, transfer... 

SDS PAGE, see electrophoresis Selective immune tolerance, 7 Semi-dry blotting, 236 Sepharose, 33, 274 Sequential epitope, 256 Serum, 35... 

For semi-dry blotting it is possible to use discontinuous buffer and/or elution promoting buffer on the gel side and a retention promoting buffer on the membrane side (37). 

Semi-dry blotting apparatus PVDF membrane (Thermo Scientific)... 

For western blotting procedure the proteins were transferred from the gels to nitrocellulose papers by elektro transfer with a semi dry blotting system. Immunedetection of ELIP was carried out in general as described by Towbin et al (8) with a horse-radish-peroxidase-conjugated secondary antibody to visualize the antigen-antibody-complex on the nitrocellulose sheets. 

Fig. 3 Detection of BLIP after different periods of illumination. Pea seedlings were grown in total darkness for six days and then illuminated as indicated. Thylakoid membrane proteins were isolated, resolved on a 12.5 % SDS-gel and transferred to a nitrocellulose sheet by a semy dry blotting system. BLIP was immunodetected with an BLIP specific primary antibody and a horse radish peroxidase conjugated secondary antibody.

The Bio-Rad Trans-Blot Semi-Dry system is used for Western blotting. 

Vertical electrophoresis systems (Amersham Pharmacia Biotech, Piscataway, NJ, USA) Trans-Blot semi-dry transfer cell (Bio-Rad, Hercules, CA, USA) hybridization incubator (Fisher, Hanover Park, IL, USA) personal densitometer SI (Molecular Dynamics, Sunnyvale, CA, USA). Additional materials and equipment needed are described throughout the text. All chemicals are of reagent grade. 

All sample proteins were run on either 12% or 14% pre-cast Novex 1.0 mm, 10 well gels under non-reducing conditions according to Laemmli (7). Samples were immediately electroblotted to Immobilon-P PVDF membrane using a semi-dry (MilliBlot-SDE) electroblotter essentially quantitiatively (8). After blotting, PVDF was washed briefly in HPLC water and stained with 0.05% Brilliant Blue-G Coomassie (BB-G) /20% methanol /0.5% acetic acid or Amido Black (2). The membrane was kept wet and not allowed to dry (9). Enzymatic digestion was performed as described (2) with all digestion and extraction buffer volumes reduced to 25 pL. 

This protocol is intended to use the Bio-Rad Mini PROTEAN 3 cell and Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell. Any other apparatus would work well under similar conditions. 

Alfred Nobel Drive Hercules, CA 94547, USA PHONE (800) 424-6723 FAX (800) 879-2289 [Trans-Blot SD 170-3940 (semi-dry) Trans-Blot 170-3939]... 

Electroblotting is the most commonly used method of transferring proteins from a gel to a membrane. The principal advantages are the speed and the completeness of transfer compared with diffusion or vacuum blotting. Electroelution can be achieved either by (1) complete immersion of a gel-membrane sandwich in a buffer (wet transfer) or (2) placing the gel-membrane sandwich between absorbent paper soaked in transfer buffer (semi-dry transfer). 

Semi-dry transfer For the semi-dry transfer, the gel-membrane sandwich is placed between carbon plate electrodes. Semi-dry or horizontal blotting uses two plate electrodes (stainless steel or graphite/ carbon) for a uniform electrical field over a short distance, and sandwiches between these of up to six gel/membrane/filter paper assemblies, all well soaked in transfer buffer. The assembly is clamped or otherwise secured on its side and electrophoretic transfer effected in this position, using as transfer buffer only the liquid contained in the gel and filter papers or other pads in the assembly. 

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