![]() Place the membrane in blocking buffer and incubate at room temperature for 1 hour (or at 4☌ overnight) with shaking. Of the antigen by HRP reaction) ※An example performed at MBL Probing with antibodies and detection of the bands (blocking and detection Ponceau S is commonly used for this purpose because the membrane is easily destained, and it does not interfere with subsequent probing with antibodies. Turn off the power supply, remove the membrane, and temporarily stain to check the efficiency of transfer and to visualize the molecular weight markers to confirm their positions. *Transfer conditions are different depending on experimental conditions. Turn on the power supply and transfer for 1 hour at 50 mA (for one gel)*. Place a piece of equilibrated filter paper on the anode plate, and place the membrane, gel, and another piece of filter paper without any air bubbles.Ĭonnect the plus electrode to the anode plate and connect the negative electrode to the cathode plate. Skip using methanol, and equilibrate the membrane in transfer buffer.Įquilibrate the gel in transfer buffer after electrophoresis. ※There is no need for pre-wetting a nitrocellulose membrane. Soak a piece of PVDF membrane slightly larger than the gel in methanol for 1 minute (pre-wetting), and then equilibrate in transfer buffer.Īlso equilibrate two pieces of filter paper slightly larger than the PVDF membrane in transfer buffer. Transfer of proteins to a membrane (semi-dry type blotting and membrane staining)
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