Recently, I got a problem on western blot.
Smaples are not apart as molecular size, and even marker is not apart from about 70kb to 130kb.
I did make a new gel buffer twice, changing tris and 10% SDS.
But it doesn't work.
Samples are not aprat, yet.
I guess arcylamide got a problem. For example, mixed with somthing...
Did who get a problem like this? plz give me a some clue.
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Thanks to everybody answer me :) I did change a Arcylamide, Low gel buffer(of course Tris and SDS, respectively), and Running buffer. But it didn't work. Especitally, i made a low percentage Acrylamide gel, it separates 70~130kDa well. But it aggregates 7~40kDa. So that could be a solution.
I solved this problem by changing a new TEMED. We used one TEMED for about 3 years. Finally, it separates markers and samples well.
Thank you for your guys' favor
Hi Menseok
In your case the possible reason seam to be the running time of the gel.
how long you run your gel?
Hi
You should prepare fresh Acrylamide, may be something mixed up and use 8% gel.
Good Luck !!
Hi Minseok,
If i get you right,its like your problem is the gel running. I will suggest you use 8-10% SDS-PAGE and run at 100V till the dye front reach the bottom of the gel. Ensure that you check the followings(1)Your running buffer composition(2)Acrylamide(is it new or old)-use freshly prepared acrylamide.
I hope this will help you.
Hello Minseok,
Are you facing problem during the transfer of proteins from 70kDa to 130kDa or during separation in SDS-PAGE??
Try to make 7.5% SDS-PAGE and run your sample for a longer time at 80V, provided you don't need lower molecular weight proteins (
Thanks to everybody answer me :)
I did change a Arcylamide, Low gel buffer(of course Tris and SDS, respectively), and Running buffer. But it didn't work.
Especitally, i made a low percentage Acrylamide gel, it separates 70~130kDa well. But it aggregates 7~40kDa. So that could be a solution.
I solved this problem by changing a new TEMED.
We used one TEMED for about 3 years.
Finally, it separates markers and samples well.
Thank you for your guys' favor
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