1 (lysate sample failed) 4-11(purified protein samples worked)
Purified and lysate samples are loaded and should contain S. cerevisiae Rpn11(homolog of PSMD14). Lysate samples showed no signal, but all purified proteins appear at the correct molecular weight (MW). non-specific bands were not seen in any samples.
After development with the ab109123 (top), blot was stripped reblotted against M2-FLAG (bottom). For lanes 4 and 5, FLAG signal was not present, as expected, but bands of the same MW as the antiPSMD14 samples were present for lanes 6-11. No signal was observed in lanes 1 and 2, which was unexpected for lane 1. Lower MW species seen for lane 6, and a ~100kDa band seen in lane 6 were also present in the anti FLAG blot, therefore it is likely that those bands are truncated or modified Rpn11 not non-specific bands.
1: 3uL of S. cerevisiae lysate expressing Rpn11 (H109A, H111A)-FLAG
2: 3uL of the above lysate after FLAG IP
3: ladder (not visible in Chemiluminescence)
4: Rpn11 residues 1-239 untagged, Recombinantly expressed in E. coli, purified protein that forms a dimer with Rpn8. MW: 27kDa
5: Rpn11, untagged, Recombinantly expressed in E. coli, purified protein as proteasome 'lid' complex MW: 34kDa
6: Rpn11, with C-terminal FLAG-HIS tag, endogenous purified protein, purified as proteasome 'lid' MW: 38kDa, sample loaded with 3x the protein of other lanes
7: Rpn11 (H109A, H111A), with C-terminal FLAG-HIS tag, endogenous purified as proteasome 'lid' complex. MW: 36kDa.
8, 9: Rpn11 (H109A, H111A), with C-terminal FLAG-HIS tag, endogenous purified protein as 26S proteasome holoenzyme complex MW: 36kDa.
10, 11: Rpn11 (H109A, H111A), with C-terminal FLAG-HIS tag,endogenous purified protein as proteasome 'lid' complex purified protein and independent prep from #8 & #9 protein. MW: 36kDa. ~1/3 as much protein as other samples (excluding #6)
Submitted Aug 01 2017
Get resources and offers direct to your inboxSign up