A basic Western blot question - (Mar/26/2008 )
Hi,
I am wondering which direction should the gel run to transfer the protein from the gel to the membrane? I know that from anode to cathode we have filter, gel, membrane and filter again. My question is that does it matter which side of the gel is in contact with the membrane?
My other question is about running the gel. After putting the gel in the tank, do you fill both upper and lower tank with running buffer before loading the sample or you fill the lower tank after loading the sample?
Thanks
-sayeh-
QUOTE (sayeh @ Mar 26 2008, 10:25 AM)
Hi,
I am wondering which direction should the gel run to transfer the protein from the gel to the membrane? I know that from anode to cathode we have filter, gel, membrane and filter again. My question is that does it matter which side of the gel is in contact with the membrane?
My other question is about running the gel. After putting the gel in the tank, do you fill both upper and lower tank with running buffer before loading the sample or you fill the lower tank after loading the sample?
Thanks
I am wondering which direction should the gel run to transfer the protein from the gel to the membrane? I know that from anode to cathode we have filter, gel, membrane and filter again. My question is that does it matter which side of the gel is in contact with the membrane?
My other question is about running the gel. After putting the gel in the tank, do you fill both upper and lower tank with running buffer before loading the sample or you fill the lower tank after loading the sample?
Thanks
It shouldn't matter which side is in contact, although it might help if you want to keep your orientation a certain way (such as ladder always to the left, etc.). I always load my sample first and then add buffer to the bottom simply because i want to monitor the top to see if it's leaking, and it helps to be able to see if any buffer is coming out the bottom. Otherwise, I don't think that it should matter.
-smu2-
QUOTE (smu2 @ Mar 26 2008, 12:32 PM)
QUOTE (sayeh @ Mar 26 2008, 10:25 AM)
Hi,
I am wondering which direction should the gel run to transfer the protein from the gel to the membrane? I know that from anode to cathode we have filter, gel, membrane and filter again. My question is that does it matter which side of the gel is in contact with the membrane?
My other question is about running the gel. After putting the gel in the tank, do you fill both upper and lower tank with running buffer before loading the sample or you fill the lower tank after loading the sample?
Thanks
I am wondering which direction should the gel run to transfer the protein from the gel to the membrane? I know that from anode to cathode we have filter, gel, membrane and filter again. My question is that does it matter which side of the gel is in contact with the membrane?
My other question is about running the gel. After putting the gel in the tank, do you fill both upper and lower tank with running buffer before loading the sample or you fill the lower tank after loading the sample?
Thanks
It shouldn't matter which side is in contact, although it might help if you want to keep your orientation a certain way (such as ladder always to the left, etc.). I always load my sample first and then add buffer to the bottom simply because i want to monitor the top to see if it's leaking, and it helps to be able to see if any buffer is coming out the bottom. Otherwise, I don't think that it should matter.
Thanks a lot
-sayeh-