Bio-Rad Western Blot半干法转膜的十大注意事项

Bio-Rad Western Blot半干法转膜的十大注意事项 Do not immerse the unit in liquid. Use special care when cleaning the anode plate to avoid scratching or marring the platinum. Do not use abrasives or strong detergents. The cathode plate (stainless steel) c an be cleaned with a mild abrasive to remove salt that may deposit du ring normal operation. The entire unit can also be periodically disas sembled and cleaned with water to remove salt deposits. 推荐清洗方法:转膜结束后,立即用双蒸水将转膜仪冲洗几次,并晾干。 Electrophoretic transfer of proteins and nucleic acids is dependent o n many factors. Observe the following guidelines to avoid mishaps tha t may result in serious damage to the instrument or injury to the ope rator. 1. Do not reverse polarity on this instrument. This will result in c orrosion and rusting of the stainless steel cathode. If this should o ccur, the stainless steel should be cleaned with a mild abrasive clea ner to remove the rust. 2. Do not exeed 25 V with this instrument. This could damage the ele ctrodes. 3. Do not adjust the pH of transfer buffers unless specifically indi cated. Following instructions carefully. Adjustment of pH of transfer buffers, when not indicated, will result in increased buffer conduct ivity. This is manifested by a higher than expected initial current o utput as shown by the power supply's current meter. Monitor buffer re sistance with the Model 200/2.0 power supply prior to each run to ins ure proper buffer conductivity. 4. Lengthy tranfer times are not recommended. Do not leave this instr ument unattached. Joule heat can be generated rapidly during semi-dry blotting. Transferring longer than 2 hours can damage the unit. 5. Power supply requirements. The Trans-Blot SD cell should only be u sed with the microprocessor-controlled Model 200/2.0 power supply or the Model 1000/500 power supply. 6. Do not operate this instrument in ambient temperatures exceeding 5 0 ℃. 7. Buffer preparation is extremely important. Do not adjust transfer buffer pH by addition of acid or base unless specifically indicated in the instructions. Improperly prepared buffer will cause excess hea t generation and safety hazards. Use only high quality, reagent grade methanol. Contaminated methanol can result in increased transfer buf fer conductivity, as well as poor transfer of macromolecules. 8. Following electrophoresis, equilibrate the gels in transfer buffe r. Equilibration facilitates the removal of electrophoresis buffer sa lts and detergents. If the salts are not removed, they will increase the conductivity of the transfer buffer and the amount of heat genera ted during the transfer. Also, low percentage gels (<12% acrylamide) will shrink in methanol-containing buffers. Equilibration allows the gel to adjust to its final size prior to electrophoretic transfer. T he length of time required for equilibration is dependent on the gel thickness. For example, 15 minutes for a 0.75 mm SDS-PAGE gel. Low molecular weight macromolecules ( 10,000 daltons) may diffuse ou t of gels more readily. One can allow adequate gel pre-equilibration by changing the pre-equilibration buffer several times during a relat ively short pre-equilibration period. This will help to limit diffusi on of low molecular weight macromolecules while providing efficient s alt reduction. 9. Cut the membrane to the dimensions of the gel. Wet the membrane by slo wly sliding it at a 45° angle into transfer buffer and allowing it to soak for 15–30 minutes. Complete wetting of the membrane is im portant to insure proper binding. Abrupt wetting can lead to entrapme nt of air bubbles in the matrix. These air bubbles can block transfer of molecules. To avoid membrane contamination, always use forceps o r wear gloves when handling membranes. 10. Cut filter paper to the dimensions of the gel. Two pieces of extr a thick filter paper (or four pieces of thick or six pieces of thin filter paper) per gel are needed for each gel/membrane sandwich. Com pletely saturate the filter paper by soaking in transfer buffer. 继续阅读