Hope everyone is well.. I just have a quick question and any response would be greatly appreciated!! ..
I am in the process of expressing a fickly flagellin protein on a larger scale to get antibodies. We have cloned into PgMET and then into PQE-Xa 30 we transformed into DHAlpha 5 cells and all constructs are perfect... We then induced with IPTG (at 1 and 3 hrs) at 1 MM, then ran an SDS Page and a Western Blot on a small scale (USING ONLY PELLET AND NOT SUPERNATE) and it worked perfectly.
However, when I did this this on a larger scale and am running into some trouble:
Induction we decided to go with 1hr and 3hr at 1 mm with IPTG in which we only use the supernate from purification: using NI/NTA in column: Wash (2 washes) /Eluding (4 washes)both with a Ph of 8. For SDS Page we run the samples at a quantitiy of 20 ul at 150 v for an hour.
Western Blot Conditions:
1 primary Antibody ratio (1:5000) 1 ul Penta his tag antibody in 5 ml
No milk only blocking buffer
On the second go we keep induction at 1hr/1mm with IPTG I have altered the PH (wash buffers 8.0, 7.4, 6.3, 5.9 then eluding 4 times at 5.9 PH from the previous of both buffers being a PH of 8) using a quantity of 40 ul SDS Page bands are there and in better shape than previous SDS-Page however still faint and GENE SNAP cannot pick them up so I have no picture, and there is still nothing on the Western Blot...
I can only think of the following that could be going wrong:
- His-tag is hiding (however I would think this is big enough this would not happen)/ something with anti body?
- PH needs to be altered a little more
- The protein is insoluble, partially insoluble, soluble but in cell fragments (perform solubility test) However, it is a flagellin protein which would make no sense why it would be insoluble unless it’s just a building block or something of the sort.
I am not sure if anyone could think of anything else or has any suggestions but it would be greatly appreciated!
Edited by London28, 19 July 2010 - 07:51 AM.