Western Blots with (250 kDa) High Molecular Weight Protein - (Jul/25/2007 )
Hi,
I have been doing western blots with for proteins less than 150 kDa, and have a new antibody for a protein that is 250 kDa. I can not seem to get this protein to run in far enough without melting the gel. Usually with the lower MW protenis I use either a 10 or 12 % PAGE gel or a premade 4-12% gradient NuPAGE gel. I usually run the gel 100 V 1.5 hrs, but tried first 100 V 2.5 hrs, then 50 mAmp 2 hrs on an 8% gel as well as a 4-12% gradient gel. None of these results look good- they are either sloppy (melty-gel looking) or nothing is even there.
I have heard that transfering longer will help, I tried also, still no good. The antibody is anti-Gakin (KIF13B) and I am using it in lysate from total brain, as well as Oligodendrocytes and Neurons. Any ideas? OR even better a protocol for higher MW proteins?
Thanks!
well my protein is 2254aa and runs at 250kD. I do 6%SDS page. I use a separating gel of roughly 7,5-8cm and the stacking is 0.5 to1cm.
The full length of the protein is at 1cm in resolving at most.
So 10 or 12%... well seem to concentrate for me.
I run the gel at 100V till the blue dye reach the end. But i'm sure it's for at least 2h. Alternatively, i run at 80V, pick up tha mA value. I stop the running and goes for constant mA, with the previously picked value. Work also nice.
I transfered on semi dry apparatus. No ignal with 8h transfert. Nice with 24h transfert and a 2overnight Ist ab incubation.
But my colleague did have success with 1overnight incubation. Secondary antibody, 2h RT
The full length of the protein is at 1cm in resolving at most.
So 10 or 12%... well seem to concentrate for me.
I run the gel at 100V till the blue dye reach the end. But i'm sure it's for at least 2h. Alternatively, i run at 80V, pick up tha mA value. I stop the running and goes for constant mA, with the previously picked value. Work also nice.
I transfered on semi dry apparatus. No ignal with 8h transfert. Nice with 24h transfert and a 2overnight Ist ab incubation.
But my colleague did have success with 1overnight incubation. Secondary antibody, 2h RT
I have been runing WB for a 460 kDa phosphop-protein and I use 5% SDS mini gel with 4% steaking (1 cm). I run the gel at 100 V until the 75 kDa band reaches de bottom of the gel. I transfers o/n at 35 V, 4 C, then 1 h at 100 V next morning, 4 C (Thanks Minnie Mouse and Minkewi). The transfer buffer recipe: 48 mM Tris base, 39 mM Glycine, 0.04 % SDS, 10% methanol.
This works excellent for me.