Problems with blot - (Nov/03/2008 )
I have been having this problem repeatedly. When I transfer two gels, the membrane farthest away from the ice block has splotches on it after ponceau staining while the other blot looks fine and has nice protein transfer. The membrane with the splotches also has some protein but not where the splotches are. I noticed today that when I tried to run the transfer at 100V it only went up to 91 V. I have tried to use cold transfer buffer and have remade the transfer buffer. I have also tried using a different power supply but with the same results. Is it the buffer or is there something else I should do? We use methanol when making our 1x transfer buffer and we use nitrocellulose membranes. If you have any suggestions that would be great! Thanks!
-koralreef3516-
QUOTE (koralreef3516 @ Nov 3 2008, 02:48 PM)
I have been having this problem repeatedly. When I transfer two gels, the membrane farthest away from the ice block has splotches on it after ponceau staining while the other blot looks fine and has nice protein transfer. The membrane with the splotches also has some protein but not where the splotches are. I noticed today that when I tried to run the transfer at 100V it only went up to 91 V. I have tried to use cold transfer buffer and have remade the transfer buffer. I have also tried using a different power supply but with the same results. Is it the buffer or is there something else I should do? We use methanol when making our 1x transfer buffer and we use nitrocellulose membranes. If you have any suggestions that would be great! Thanks!
I can make a few suggestions. First, make sure you have a magnetic stir bar in your tank. Stirring your transfer buffer during your transfer will make sure that the ice block is able to cool all of the transfer buffer and not just the area around the block.
Also, make sure that your amp and watt settings on your power supply are not limiting. I don't recall the specific numbers, but when I transfer at 100V, my amps and watts are really high.
Finally, try wiping down the wire in your transfer aparatus gently with a gloved finger. Sometimes gunk can build up on that and inhibit current flow.
Good luck!
-jlolsen-
QUOTE (jlolsen @ Nov 3 2008, 05:37 PM)
QUOTE (koralreef3516 @ Nov 3 2008, 02:48 PM)
I have been having this problem repeatedly. When I transfer two gels, the membrane farthest away from the ice block has splotches on it after ponceau staining while the other blot looks fine and has nice protein transfer. The membrane with the splotches also has some protein but not where the splotches are. I noticed today that when I tried to run the transfer at 100V it only went up to 91 V. I have tried to use cold transfer buffer and have remade the transfer buffer. I have also tried using a different power supply but with the same results. Is it the buffer or is there something else I should do? We use methanol when making our 1x transfer buffer and we use nitrocellulose membranes. If you have any suggestions that would be great! Thanks!
I can make a few suggestions. First, make sure you have a magnetic stir bar in your tank. Stirring your transfer buffer during your transfer will make sure that the ice block is able to cool all of the transfer buffer and not just the area around the block.
Also, make sure that your amp and watt settings on your power supply are not limiting. I don't recall the specific numbers, but when I transfer at 100V, my amps and watts are really high.
Finally, try wiping down the wire in your transfer aparatus gently with a gloved finger. Sometimes gunk can build up on that and inhibit current flow.
Good luck!
We have always used a stir bar when running and I believe the amps on the last transfer was 400 mA but the volts were only 91V when I set it to 100V. Is there something wrong with the buffer then? We are also having problems running our SDS-PAGE gels so it makes me feel like it is not the buffer. Could it just be air bubbles when transferring? It is just odd that it is always the blot farthest away from the ice block that has the splotches. I will try cleaning the wires in the transfer and see how that helps. Thanks for your suggestions! I really appreciate them because I am getting pretty frustrated with this.
-koralreef3516-