07x_ip SemiDry IMMUNOBLOTTING_102407


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PROTEIN BLOTTING WITH SEMIDRY SYSTEMS Even and efficient transfer of most proteins is also possible with semidry blotting, a convenient alternative to tank transfer systems. Instead of being placed vertically into a tank filled with transfer buffer, the gel is held horizontally between buffer-saturated blotting paper that is in contact with the electrodes (Fig. 8.10.2), greatly reducing the amount of buffer required. The electrodes are close together, giving high field strengths and rapid transfer with a standard electrophoresis power supply. Prolonged transfers (> 1 hr) are not recommended; tank blotting should be used for proteins that require long blotting times for efficient transfer. Additional Materials Six sheets of Whatman 3MM filter paper or equivalent, cut to size of gel and saturated with transfer buffer Semidry transfer unit (Hoefer, Bio-Rad or Sartorius) 1. Prepare samples and separate proteins using small or standard-sized one- or two-di mensional gels. Because transfer efficiency depends on many factors (e.g., gel concentration and thickness, protein size, shape, and net charge) results may vary. Below is a guideline for o. 75-mm-thick SDS-PAGE gels transferred by semidry blotting. Figure 8.10.2 Immunoblotting with a semidry transfer unit. Generally, the lower electrode is the anode, and one gel is transferred at a time. A Mylar mask (optional in some units) is put in place on the anode. This is followed by three sheets of transfer buffer-soaked filter paper, the membrane, the gel, and finally, three more sheets of buffer-soaked filter paper. To transfer multiple gels, construct transfer stacks as illustrated, and separate each with a
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This note was uploaded on 03/09/2010 for the course BIOL 371 taught by Professor Eugenemuller during the Fall '09 term at Framingham State College.

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