3 replies to this topic

[Juan Diego Prieto]

Hi everybody,

 

I've been trying to detect the chicken IP3R in a Western Blot, but since it's a kind of heavy protein, ca. 320 kDa, I've had no success. I am reviewing all the conditions and wanted to know what would be the adequate %gel, voltage and running time to discern clearly the protein in the gel. The transference onto the membrane is another step, about which I'll ask later. Please help me out! Thanks in advance.

[mdfenko]

is the protein purified or a mixture? if a mixture, do you want to see the other proteins on the blot?

 

is the protein multiple or single subunit?

 

if multiple, do you want to separate the subunits during page? if so, then what are the sizes of the subunits?

 

are you using native or sds-page?

 

if you do have to separate a 320 kDa protein then best would be to use a 5% gel but that would be very fragile and hard to work with for setting up the transfer so a good choice will be 7% (assuming 30:0.8% (acrylamide:bisacrylamide)).

 

check the manual that came with the apparatus for recommended voltage and timing.

[Juan Diego Prieto]

is the protein purified or a mixture? if a mixture, do you want to see the other proteins on the blot?

 

is the protein multiple or single subunit?

 

if multiple, do you want to separate the subunits during page? if so, then what are the sizes of the subunits?

 

are you using native or sds-page?

 

if you do have to separate a 320 kDa protein then best would be to use a 5% gel but that would be very fragile and hard to work with for setting up the transfer so a good choice will be 7% (assuming 30:0.8% (acrylamide:bisacrylamide)).

 

check the manual that came with the apparatus for recommended voltage and timing.

 

It's a mixture of proteins but I'm only interested in that one. The receptor is a complex but I'd be detecting the monomer, which is ca. 320 kDa. SDS-PAGE is the method I'm using. The apparatus' manual is nowhere to be found. Any advice on approx. voltage and running time? Thank you!

[mdfenko]

i always prefer to run with constant current and allow voltage to float (with the maximum set at 400 volts, which it never reaches). if you want to run at constant voltage then 100 volts is reasonable.

 

timing depends on the size of the apparatus. i determine the run is finished when the tracking dye (i use bromphenol blue, other tracking dyes, like phenol red, migrate at different rates) reaches the bottom of the gel.