western blot- some bands not seen - western blot (Jul/23/2007 )
Hello
I'm trying to do a western blot on certain sodium channels in the heart and I noticed that my positive control is working (GAPDH) but i cannot see a band for the sodium channels (Nav1.1, 1.3, 1.5, 1.6) . Now to tell you the nature of the sodium channels, they are huge proteins, 250 kdaltons, and there arent many of them in the heart, and so i had to be careful in the transfer, and had to transfer longer, and so I transferred overnight with 200mA...also, i loaded 50 micrograms of protein, i'm not sure if i need to load more?
i'm not sure how i should begin the trouble shooting.
also, although i see the GAPDH band, it is very messy. i blocked for an hour, is this enough?
thank you!!
-westernblot-
QUOTE (westernblot @ Jul 23 2007, 10:00 PM)
Hello
I'm trying to do a western blot on certain sodium channels in the heart and I noticed that my positive control is working (GAPDH) but i cannot see a band for the sodium channels (Nav1.1, 1.3, 1.5, 1.6) . Now to tell you the nature of the sodium channels, they are huge proteins, 250 kdaltons, and there arent many of them in the heart, and so i had to be careful in the transfer, and had to transfer longer, and so I transferred overnight with 200mA...also, i loaded 50 micrograms of protein, i'm not sure if i need to load more?
i'm not sure how i should begin the trouble shooting.
also, although i see the GAPDH band, it is very messy. i blocked for an hour, is this enough?
thank you!!
I'm trying to do a western blot on certain sodium channels in the heart and I noticed that my positive control is working (GAPDH) but i cannot see a band for the sodium channels (Nav1.1, 1.3, 1.5, 1.6) . Now to tell you the nature of the sodium channels, they are huge proteins, 250 kdaltons, and there arent many of them in the heart, and so i had to be careful in the transfer, and had to transfer longer, and so I transferred overnight with 200mA...also, i loaded 50 micrograms of protein, i'm not sure if i need to load more?
i'm not sure how i should begin the trouble shooting.
also, although i see the GAPDH band, it is very messy. i blocked for an hour, is this enough?
thank you!!
Do you see the marker for those sizes? That will let you know if your transfer conditions are right. You may need to load more protein - for large rare proteins I load up to 80ug but that is on a maxi-gel. If you are using a mini-gel I don't know if you can load that much. What do you mean about the GAPDH being messy? Is there a lot of background or is the band not straight and clear? Blocking for one hour at room temp is usually enough but that depends on your blocking solution, which you can change to try and get better results.
-syaniv-