In lane D (can not cleave itself) I do see a strong band, albeit at a lower MW then expected, but I think it is right.
In lane E I should observe cleaved proteins (5-6) and a few intermediates, instead I get a smear with only one band visible (about 1/3 down from the top)
I ran a 4-20% HEPES gel from Pierce. I use SDS-HEPES buffer. I have treated all samples with 2X beta mercaptoethanol SDS loading buffer, heated at 70C for 10 minutes, and ran for 2 hrs at 30 milliamps. I exposed for 90 minutes (overnight does not show additional bands)
Can anybody explain the smears? I should be seeing bands from 20-60 Kd in lane E (possibly intermediates at higher MW).
I used the TnT Quick Coupled transcription/translation kit from Promega. The lysate is very viscous and is difficult to run on gels. If anybody has experience with the Promega TnT kit, please let me know.
In short, I really need to improve the quality of these gels with better resolution. My constructs have been sequenced and they should be fine for the TnT reaction. There should be one band in lane D, and around 6 bands in lane E. I am hoping the additional smears in lane E contain the desired proteins and I am simply running the gels incorrectly, somehow.
Thanks for any advice.
Edited by HOYAJM, 15 March 2013 - 12:34 PM.